Diagnostics and therapeutics for cardiovascular disease

ABSTRACT

The kits and methods of the present invention relate to the diagnosis of cardiovascular disorders. In one aspect, the invention discloses a method and a kit for determining whether a subject has a fragile plaque disorder. In one aspect, the invention discloses a method and a kit for determining whether the subject has an occlusive disorder. In one aspect, the invention discloses a method and a kit for determining whether the subject has a restenosis disorder. Other methods of the present invention relate to the selection of therapeutics for a patient with a cardiovascular disease.

RELATED U.S. APPLICATIONS

[0001] The present application is a continuation-in-part of U.S.application Ser. No. 09/320,395, filed May 26, 1999, which applicationis hereby incorporated by reference. The present application furtherclaims benefit of priority to U.S. application Ser. No. 08/813,456,filed Mar. 10, 1997.

TECHNICAL FIELD

[0002] The present invention relates to kits and methods for thediagnosis and treatment of cardiovascular disorders, and morespecifically to kits and methods related to diagnosis of disordersassociated with IL-1 genotype patterns.

BACKGROUND OF THE INVENTION

[0003] Atherosclerosis (or arteriosclerosis) is the term used todescribe progressive luminal narrowing and hardening of the arteriesthat can result in an aneurysm, ischemia, thrombosis, embolism formationor other vascular insufficiency. The disease process can occur in anysystemic artery in the human body. For example, atherosclerosis in thearteries that supply the brain (e.g. the carotids, intracerebral, etc.,)can result in stroke. Gangrene may occur when the peripheral arteriesare blocked, and coronary artery disease occurs when the arteries thatsupply oxygen and nutrients to the myocardium are affected.

[0004] Coronary artery disease is a multifactorial disease that resultsin the deposition of atheromatous plaque and progressive luminalnarrowing of the arteries that supply the heart muscle. Theatherosclerosis process involves lipid induced biological changes in thearterial walls resulting in a disruption of homeostatic mechanisms thatkeeps the fluid phase of the blood compartment separate from the vesselwall. Since the normal response to all injury is inflammation, theatherosclerotic lesion shows a complex chronic inflammatory response,including infiltration of mononuclear leukocytes, cell proliferation andmigration, reorganization of extracellular matrix, andneovascularization. In fact, the atheromatous plaque consists of amixture of inflammatory and immune cells, fibrous tissue, and fattymaterial such as low density lipids (LDL) and modifications thereof, andα-lipoprotein. The luminal narrowing or blockage results in reducedability to deliver oxygen and nutrients to the heart muscle, producingmyocardial infarction, angina, unstable angina, and sudden ischemicdeath as heart failure. Though occlusion usually progresses slowly,blood supply may be cut off suddenly when a portion of the built-uparterial plaque breaks off and lodges somewhere in an artery to block ittemporarily, or more usually, when thrombosis occurs within the arteriallumen. Rupture of the fibrous cap overlaying a vulnerable plaque is themost common cause of coronary thrombosis. Depending on the volume ofmuscle distal to the blockage during such an attack, a portion of themyocardial tissue may die, weakening the heart muscle and often leadingto the death of the individual.

[0005] For many years, the most common measure of imminent risk for aheart disease “clinical event”, such as a myocardial infarction ordeath, was physical blockage of the coronary arteries, as assessed bytechniques such as angiography. During the early 80's studies by DeWoodand coworkers (N. Engl. J. of Med. (1980) 303:1137-40), revealed thatocclusive thrombus was responsible for most cases of acute myocardialinfarction. At that time, the prevailing concept was that myocardialinfarction resulted from occlusion at a site of high grade stenosis. In1988, Little et al. (Circulation (1988) 78:1157-66), showed most of theinfarctions resulted from a coronary blockage that had previously showna stenosis of less than 50% on angiography. Therefore, the severity ofthe coronary stenosis did not accurately predict the location of asubsequent coronary blockage. With these studies the importance ofvulnerable atherosclerotic plaque became evident.

[0006] It is now clear that rupture at the site of a vulnerableartherosclerotic plaque is the most frequent cause of acute coronarysyndromes. Such plaque does not cause high grade stenosis, but mayresult in acute coronary syndrome, such as unstable angina, myocardialinfarction, or sudden death. No methods are currently available that canreliably identify plaques prone to rupture. In fact, development ofclinically useful imaging techniques for identifying vulnerable plaquesis an active area of research. Some of the methods are being used toidentify such plaques include for example, thermography (atheroscleroticplaques show thermal heterogeneity), spectroscopy (used to quantify theamount of cholesterol, cholesterol esters, triglycerides, phospholipidsand calcium salts present in small volumes of the coronary arterialtissue), radioisotope scintigraphy (various constituents of vulnerableplaques such as inflammatory cells may be imaged with radioisotopetechniques), and detection of inflammatory serum markers such asC-reactive protein levels.

[0007] Arterial sites that show acute plaque rupture are characterizedby chronic inflammatory components that are not found, or are at muchlower levels, in arterial plaques that are stable and unlikely to causeclinical events (Ross R. The pathogenesis of atherosclerosis: aperspective for the 1990s. Nature 1993;362:801-809.) (Libby P. Molecularbasis of the acute coronary syndromes. Circulation 1995;91:2844-2850).The current published clinical data from many sources clearlydemonstrate that various components of inflammation are strongindependent influences on the severity and clinical outcomes of coronaryartery disease (Ross R. The pathogenesis of atherosclerosis: aperspective for the 1990s. Nature 1993;362:801-809.) (Libby P. Molecularbasis of the acute coronary syndromes. Circulation 1995;91:2844-2850).In addition, laboratory work has shown that pro-inflammatory mediatorsare critical elements in the atherosclerosis process (Ross R. Thepathogenesis of atherosclerosis: a perspective for the 1990s. Nature1993;362:801-809.) (Libby P. Molecular basis of the acute coronarysyndromes. Circulation 1995;91:2844-2850).

[0008] The causes and mechanisms of the atheromatous plaque build-up arenot completely understood, though many theories exist. One theory on thepathogenesis of atherosclerosis involves the following stages: (1)endothelial cell dysfunction and/or injury, (2) monocyte recruitment andmacrophage formation, (3) lipid deposition and modification, (4)vascular smooth muscle cell proliferation, and (5) synthesis ofextracellular matrix. According to this theory, the initiation ofatherosclerosis is potentially due to a form of injury, possibly frommechanical stress or from chemical stress. How the body responds to thisinjury then defines whether, and how rapidly, the injury deterioratesinto an atherosclerotic lesion. This, in turn, can result in arterialluminal narrowing and damage to the heart tissue which depends on theblood flow of oxygen and nutrients.

[0009] For many years, epidemiologic studies have indicated that anindividual's genetic composition is a significant risk factor fordevelopment of a vascular disease. For example, a family history ofheart disease is associated with an increased individual risk ofdeveloping coronary artery disease. Lipid and cholesterol metabolismhave historically been considered the primary genetic influence oncoronary artery disease. For example, deficiency in cell receptors forlow-density lipids (LDL), such as in familial hypercholesterolemia, isassociated with high levels of plasma LDL and premature development ofatherosclerosis (Brown & Goldstein, Sci., 191 (4223):150-4 (1976)).

[0010] Inflammation is now generally regarded as an important componentof the pathogenic process of atherosclerosis (Munro, Lab Invest.,58:249-261 (1988); Badimon, et al., Circulation, 87:3-16 (1993); Liuzzo,et al., N.E.J.M., 331(7):417-24 (1994); Alexander, N.E.J.M.,331(7):468-9 (1994)). Damage to endothelial cells that line the vesselsleads to an accumulation of inflammatory cytokines, including IL-1,TNFα, and the release of prostanoids and growth factors such asprostaglandin I₂ (PGI₂), platelet-derived growth factor (PDGF), basicfibroblast growth factor (bFGF), and granulocyte-monocyte cellstimulating factor (GM-CSF). These factors lead to accumulation andregulation of inflammatory cells, such as monocytes, that accumulatewithin the vessel walls. The monocytes then release additionalinflammatory mediators, including IL-1, TNF, prostaglandin E₂, (PGE₂),bFGF, and transforming growth factors α and β (TGFα, TGFβ). All of theseinflammatory mediators recruit more inflammatory cells to the damagedarea, regulate the behavior of endothelial and smooth muscle cells andlead to the accumulation of atheromatous plaques.

[0011] Several inflammatory products, including IL-1β, have beenidentified in atherosclerotic lesions or in the endothelium of diseasedcoronary arteries (Galea, et al., Ath. Thromb. Vasc. Biol., 16:1000-6(1996)). Also, serum concentrations of IL-1β have been found to beelevated in patients with coronary disease (Hasdai, et al., Heart,76:24-8 (1996)). Although it was historically believed that the presenceof inflammatory agents was responsive to injury or monocyte activation,it is also possible that an abnormal inflammatory response may becausative of coronary artery disease or create an increasedsusceptibility to the disease.

[0012] A key problem in treating vascular diseases is proper diagnosis.Often the first sign of the disease is sudden death. For example,approximately half of all individuals who die of coronary artery diseasedie suddenly, Furthermore, for 40-60% of the patients who are eventuallydiagnosed as having coronary artery disease, myocardial infarction isthe first presentation of the disease. Unfortunately, approximately 40%of those initial events go unnoticed by the patient. It is now believedthat, identification and stabilization of vulnerable plaques is animportant element in the treatment of coronary atherosclerosis.Identification of the haplotype patterns in various subjects would allowin the management of cardiovascular disorders and treatment could beaimed at plaque stabilization rather than revascularization and othermore invasive methods. This is especially important, because, forvarious reasons, the perception of symptoms by the patient does notcorrelate well with the total burden of coronary artery disease(Anderson & Kin, Am. Heart J., 123(5):1312-23 (1992)).

[0013] Percutaneous transluminal coronary angioplasty (PTCA) is used totreat obstructive coronary artery disease by compressing atheromatousplaque to the sides of the vessel wall. PTCA is widely used with aninitial success rate of over 90%. However, the long-tern success of PTCAis limited by intraluminal renarrowing or restenosis at the site of theprocedure. This occurs within 6 months following the procedure inapproximately 30% to 40% of patients who undergo a single vesselprocedure and in more than 50% of those who undergo multivesselangioplasty.

[0014] Stent placement has largely supplanted balloon angioplastybecause it is able to more widely restore intraluminal dimensions whichhas the effect of reducing restenosis by approximately 50%. Ironically,stent placement actually increases neointimal growth at the treatmentsite, but because a larger lumen can be achieved with stent placement,the tissue growth is more readily accomodated, and sufficient luminaldimensions are maintained, so that the restenosis rate is nearly halvedby stent placement compared with balloon angioplasty alone.

[0015] The pathophysiological mechanisms involved in restenosis are notfully understood. While a number of clinical, anatomical and technicalfactors have been linked to the development of restenosis, at least 50%of the process has yet to be explained. However, it is known thatfollowing endothelial injury, a series of repair mechanisms areinitiated. Within minutes of the injury, a layer of platelets and fibrinis deposited over the damaged endothelium. Within hours to days,inflammatory cells begin to infiltrate the injured area. Within 24 hoursafter an injury, vascular smooth muscle cells (SMCs) located in thevessel media commence DNA synthesis. A few days later, these activated,synthetic SMCs migrate through the internal elastic lamina towards theluminal surface. A neointima is formed by these cells by their continuedreplication and their production of extracellular matrix. An increase inthe intimal thickness occurs with ongoing cellular proliferation matrixdeposition. When these processes of vascular healing progressexcessively, the pathological condition is termed intimal hyperplasia ormyointimial hyperplasia. The biology of vascular wall healing implicatedin restenosis therefore includes the general processes of wound healingand the specific processes of myointimal hyperplasia. Inflammation isgenerally regarded as an important component in both these processes.(Munro and Cotran (1993) Lab. Investig. 58:249-261; and Badimnon et al.(1993), Supp II 87:3-6). Understanding the effects of acute and chronicinflammation in the blood vessel wall can thus suggest methods fordiagnosing and treating restenosis and related conditions.

[0016] In its initial phase, inflammation is characterized by theadherence of leukocytes to the vessel wall. Leukocyte adhesion to thesurface of damaged endothelium is mediated by several complexglycoproteins on the endothelial and neutrophil surfaces. Two of thesebinding molecules have been well-characterized: the endothelialleukocyte adhesion molecule-1 (ELAM-1) and the intercellular adhesionmolecule-1 (ICAM-1). During inflammatory states, the attachment ofneutrophils to the involved cell surfaces is greatly increased,primarily due to the upregulation and enhanced expression of thesebinding molecules. Substances thought to be primary mediators of theinflammatory response to tissue injury, including interleukin-1 (IL-1),tumor necrosis factor alpha (TNF), lymphotoxin and bacterial endotoxins,all increase the production of these binding substances.

[0017] After binding to the damaged vessel wall, leukocytes migrate intoit. Once in place within the vessel wall, the leukocytes, in particularactivated macrophages, then release additional inflammatory mediators,including IL-1, TNF, prostaglandin E₂, (PGE₂), bFGF, and transforminggrowth factors α and β (TGFα, TGFβ). All of these inflammatory mediatorsrecruit more inflammatory cells to the damaged area, and regulate thefurther proliferation and migration of smooth muscle. A well-knowngrowth factor elaborated by the monocyte-macrophage is monocyte- andmacrophage-derived growth factor (MDGF), a stimulant of smooth musclecell and fibroblast proliferation. MDGF is understood to be similar toplatelet-derived growth factor (PDGF); in fact, the two substances maybe identical. By stimulating smooth muscle cell proliferation,inflammation can contribute to the development and the progression ofmyointimal hyperplasia.

[0018] Leukocytes, attracted to the vessel wall by the abovementionedchemical mediators of inflammation, produce substances that have directeffects on the vessel wall that may exacerbate the local injury andprolong the healing response. First, leukocytes activated by theprocesses of inflammation secrete lysosomal enzymes that can digestcollagen and other structural proteins. Releasing these enzymes withinthe vessel wall can affect the integrity of its extracellular matrix,permitting SMCs and other migratory cells to pass through the wall morereadily. Hence, the release of these lysosomal proteases can enhance theprocesses leading to myointimal hyperplasia. Second, activatedleukocytes produce free radicals by the action of the NADPH system ontheir cell membranes. These free radicals can damage cellular elementsdirectly, leading to an extension of a local injury or a prolongation ofthe cycle of injury-inflammation-healing.

[0019] It would be desirable to determine which patients would respondwell to invasive treatments for occlusive vascular disease such asangioplasty and intravascular stent placement. It would be furtherdesirable to identify those patients at increased risk for stenosis sothat they could be targeted with appropriate therapies to prevent,modulate or reverse the condition. It would be desirable, moreover, toidentify those individuals for whom PTCA and stent placement is asuboptimal therapeutic choice because of the risk of restenosis. Thosepatients might become candidates at earlier stages for vascularreconstructive procedures, possibly combined with other pharmacologicalinterventions.

[0020] Genetics of the IL-1 Gene Cluster

[0021] The IL-1 gene cluster is on the long arm of chromosome 2 (2q13)and contains at least the genes for IL-1α (IL-1A), IL-1β (IL-1B), andthe IL-1 receptor antagonist (IL-1RN), within a region of 430 Kb(Nicklin, et al. (1994) Genomics, 19: 382-4). The agonist molecules,IL-1α and IL-1β, have potent pro-inflammatory activity and are at thehead of many inflammatory cascades. Their actions, often via theinduction of other cytokines such as IL-6 and IL-8, lead to activationand recruitment of leukocytes into damaged tissue, local production ofvasoactive agents, fever response in the brain and hepatic acute phaseresponse. All three IL-1 molecules bind to type I and to type II IL-1receptors, but only the type I receptor transduces a signal to theinterior of the cell. In contrast, the type II receptor is shed from thecell membrane and acts as a decoy receptor. The receptor antagonist andthe type II receptor, therefore, are both anti-inflammatory in theiractions.

[0022] Inappropriate production of IL-1 plays a central role in thepathology of many autoimmune and inflammatory diseases, includingrheumatoid arthritis, inflammatory bowel disorder, psoriasis, and thelike. In addition, there are stable inter-individual differences in therates of production of IL-1, and some of this variation may be accountedfor by genetic differences at IL-1 gene loci. Thus, the IL-1 genes arereasonable candidates for determining part of the genetic susceptibilityto inflammatory diseases, most of which have a multifactorial etiologywith a polygenic component.

[0023] Certain alleles from the IL-1 gene cluster are known to beassociated with particular disease states. For example, IL-1RN (VNTR)allele 2 has been shown to be associated with osteoporosis (U.S. Pat.No. 5,698,399), nephropathy in diabetes mellitus (Blakemore, et al.(1996) Hum. Genet. 97(3): 369-74), alopecia areata (Cork, et al., (1995)J. Invest. Dermatol. 104(5 Supp.): 15S-16S; Cork et al. (1996) DermatolClin 14: 671-8), Graves disease (Blakemore, et al. (1995) J. Clin.Endocrinol. 80(1): 111-5), systemic lupus erythematosus (Blakemore, etal. (1994) Arthritis Rheum. 37: 1380-85), lichen sclerosis (Clay, et al.(1994) Hum. Genet. 94: 407-10), and ulcerative colitis (Mansfield, etal. (1994) Gastoenterol. 106(3): 637-42)).

[0024] In addition, the IL-1A allele 2 from marker −889 and IL-1B (TaqI)allele 2 from marker +3954 have been found to be associated withperiodontal disease (U.S. Pat. No. 5,686,246; Kornman and diGiovine(1998) Ann Periodont 3: 327-38; Hart and Kornman (1997) Periodontol 200014: 202-15; Newman (1997) Compend Contin Educ Dent 18: 881-4; Kornman etal. (1997) J. Clin Periodontol 24: 72-77). The IL-1A allele 2 frommarker −889 has also been found to be associated with juvenile chronicarthritis, particularly chronic iridocyclitis (McDowell, et al. (1995)Arthritis Rheum. 38: 221-28). The IL-1B (TaqI) allele 2 from marker+3954 of IL-1B has also been found to be associated with psoriasis andinsulin dependent diabetes in DR3/4 patients (di Giovine, et al. (1995)Cytokine 7: 606; Pociot, et al. (1992) Eur J. Clin. Invest. 22:396-402). Additionally, the IL-1RN (VNTR) allele 1 has been found to beassociated with diabetic retinopathy (see U.S. Ser. No. 09/037472, andPCT/GB97/02790). Furthermore allele 2 of IL-1RN (VNTR) has been found tobe associated with ulcerative colitis in Caucasian populations fromNorth America and Europe (Mansfield, J. et al., (1994) Gastroenterology106: 637-42). Interestingly, this association is particularly strongwithin populations of ethnically related Ashkenazi Jews (PCTWO97/25445).

[0025] Genotype Screening

[0026] Traditional methods for the screening of heritable diseases havedepended on either the identification of abnormal gene products (e.g.,sickle cell anemia) or an abnormal phenotype (e.g., mental retardation).These methods are of limited utility for heritable diseases with lateonset and no easily identifiable phenotypes such as, for example,vascular disease. With the development of simple and inexpensive geneticscreening methodology, it is now possible to identify polymorphisms thatindicate a propensity to develop disease, even when the disease is ofpolygenic origin. The number of diseases that can be screened bymolecular biological methods continues to grow with increasedunderstanding of the genetic basis of multifactorial disorders.

[0027] Genetic screening (also called geneotyping or molecularscreening), can be broadly defined as testing to determine if a patienthas mutations (or alleles or polymorphisms) that either cause a diseasestate or are “linked” to the mutation causing a disease state. Linkagerefers to the phenomenon wherein DNA sequences which are close togetherin the genome have a tendency to be inherited together. Two sequencesmay be linked because of some selective advantage of co-inheritance.More typically, however, two polymorphic sequences are co-inheritedbecause of the relative infrequency with which meiotic recombinationevents occur within the region between the two polymorphisms. Theco-inherited polymorphic alleles are said to be in linkagedisequilibrium with one another because, in a given human population,they tend to either both occur together or else not occur at all in anyparticular member of the population. Indeed, where multiplepolymorphisms in a given chromosomal region are found to be in linkagedisequilibrium with one another, they define a quasi-stable genetic“haplotype.” In contrast, recombination events occurring between twopolymorphic loci cause them to become separated onto distinct homologouschromosomes. If meiotic recombination between two physically linkedpolymorphisms occurs frequently enough, the two polymorphisms willappear to segregate independently and are said to be in linkageequilibrium.

[0028] While the frequency of meiotic recombination between two markersis generally proportional to the physical distance between them on thechromosome, the occurrence of “hot spots” as well as regions ofrepressed chromosomal recombination can result in discrepancies betweenthe physical and recombinational distance between two markers. Thus, incertain chromosomal regions, multiple polymorphic loci spanning a broadchromosomal domain may be in linkage disequilibrium with one another,and thereby define a broad-spanning genetic haplotype. Furthermore,where a disease-causing mutation is found within or in linkage with thishaplotype, one or more polymorphic alleles of the haplotype can be usedas a diagnostic or prognostic indicator of the likelihood of developingthe disease. This association between otherwise benign polymorphisms anda disease-causing polymorphism occurs if the disease mutation arose inthe recent past, so that sufficient time has not elapsed for equilibriumto be achieved through recombination events. Therefore identification ofa human haplotype which spans or is linked to a disease-causingmutational change, serves as a predictive measure of an individual'slikelihood of having inherited that disease-causing mutation.Importantly, such prognostic or diagnostic procedures can be utilizedwithout necessitating the identification and isolation of the actualdisease-causing lesion. This is significant because the precisedetermination of the molecular defect involved in a disease process canbe difficult and laborious, especially in the case of multifactorialdiseases such as inflammatory disorders.

[0029] Indeed, the statistical correlation between an inflammatorydisorder and an IL-1 polymorphism does not necessarily indicate that thepolymorphism directly causes the disorder. Rather the correlatedpolymorphism may be a benign allelic variant which is linked to (i.e. inlinkage disequilibrium with) a disorder-causing mutation which hasoccurred in the recent human evolutionary past, so that sufficient timehas not elapsed for equilibrium to be achieved through recombinationevents in the intervening chromosomal segment. Thus, for the purposes ofdiagnostic and prognostic assays for a particular disease, detection ofa polymorphic allele associated with that disease can be utilizedwithout consideration of whether the polymorphism is directly involvedin the etiology of the disease. Furthermore, where a given benignpolymorphic locus is in linkage disequilibrium with an apparentdisease-causing polymorphic locus, still other polymorphic loci whichare in linkage disequilibrium with the benign polymorphic locus are alsolikely to be in linkage disequilibrium with the disease-causingpolymorphic locus. Thus these other polymorphic loci will also beprognostic or diagnostic of the likelihood of having inherited thedisease-causing polymorphic locus. Indeed, a broad-spanning humanhaplotype (describing the typical pattern of co-inheritance of allelesof a set of linked polymorphic markers) can be targeted for diagnosticpurposes once an association has been drawn between a particular diseaseor condition and a corresponding human haplotype. Thus, thedetermination of an individual's likelihood for developing a particulardisease of condition can be made by characterizing one or moredisease-associated polymorphic alleles (or even one or moredisease-associated haplotypes) without necessarily determining orcharacterizing the causative genetic variation.

SUMMARY OF THE INVENTION

[0030] In one aspect, the present invention provides novel methods andkits for determining whether a subject has a cardiovascular disorder. Inone embodiment, the kits and methods of the present invention aredirected to the diagnosis of fragile plaque disorder. Diagnosis of thepresence of fragile plaque disorder identifies those patientspredisposed to the development of fragile plaque disease, characterizedby clinical events such as myocardial infarction and stroke. Diagnosingthese individuals predisposed to the development of fragile plaquedisease is especially important because the onset of the disease can beabrupt and catastrophic, without premonitory signs and symptoms.Determining which patients are at risk for developing the diseasebecause they have the disorder thus opens the possibility of earlydiagnosis of disease conditions and treating the disorder and thedisease through appropriate therapeutics.

[0031] In another embodiment, the kits and methods of the presentinvention are directed to the diagnosis of an occlusive disorder.Diagnosis of the presence of an occlusive disorder identifies thosepatients predisposed to the development of occlusive disease,characterized by clinical events such as ischemia, angina, claudication,rest pain and gangrene. Determining which patients are at risk fordeveloping the disease because they have the disorder thus opens thepossibility of early diagnosis and therapeutic intervention, at a stagebefore irreversible tissue changes have occurred in the tissues servedby the affected vessels.

[0032] In yet another embodiment, the kits and methods of the presentinvention are directed to the diagnosis of a restenosis disorder.Diagnosis of the presence of a restenosis disorder identifies thosepatients predisposed to the development of a restenosis disease,characterized by clinical events related to the recurrence of theinitial vascular stenosis that is being treated by the stent.Determining which patients are at risk for developing the diseasebecause they have the disorder thus opens the possibility of selectingtherapies for the initial vascular stenosis most likely to avoidsubsequent stenoses. Such patients might be candidates for surgicalrevascularization rather than percutaneous transluminal angioplasty, forexample, or such patients may benefit from pharmacological or topicalinterventions at an early stage that could affect the progression of therestenosis disorder.

[0033] In another aspect, the methods of the present invention providefor the treatment of a patient with a cardiovascular disorder of theabovementioned types. Treatment includes determining whether a patienthas an allelic pattern associated with a cardiovascular disorder andadministering to the patient a therapeutic adapted to the treatment ofthe cardiovascular disorder. In one embodiment, the method can includethe identification of a risk factor for the cardiovascular disorder andthe formulation of a treatment plan that reduces the effect of the riskfactor on the patient.

[0034] These and other embodiments of the present invention rely atleast in part upon the novel finding that there is an association ofpatterns of alleles at four polymorphic loci in the IL-1 gene clusterwith cardiovascular disorders. These patterns are referred to hereinpatterns 1, 2 and 3. Pattern 1 comprises an allelic pattern includingallele 2 of IL-1A (+4845) or IL-1B (+3954) and allele 1 of IL-1B (−511)or IL-1RN (+2018), or an allele that is in linkage disequilibrium withone of the aforementioned allele. In a preferred embodiment, thisallelic pattern permits the diagnosis of fragile plaque disorder.Pattern 2 comprises an allelic pattern including allele 2 of IL-1B(−511) or IL-1RN (+2018) and allele 1 of IL-1A (+4845) or IL-1B (+3954),or an allele that is in linkage disequilibrium with one of theaforementioned alleles. In a preferred embodiment, this allelic patternpermits the diagnosis of fragile plaque disorder. Pattern 3 comprises anallelic pattern including allele 1 of IL-1A (+4845) or allele 1 of IL-1B(+3954), and allele 1 of IL-1B (−511) or allele 1 of IL-1RN (+2018), oran allele that is in linkage disequilibrium with one of theaforementioned alleles. In a preferred embodiment, this allelic patternpermits the diagnosis of a restenosis disorder.

[0035] An allele associated with a cardiovascular disorder can bedetected by any of a variety of available techniques, including: 1)performing a hybridization reaction between a nucleic acid sample and aprobe that is capable of hybridizing to the allele; 2) sequencing atleast a portion of the allele; or 3) determining the electrophoreticmobility of the allele or fragments thereof (e.g., fragments generatedby endonuclease digestion). The allele can optionally be subjected to anamplification step prior to performance of the detection step. Preferredamplification methods are selected from the group consisting of: thepolymerase chain reaction (PCR), the ligase chain reaction (LCR), stranddisplacement amplification (SDA), cloning, and variations of the above(e.g. RT-PCR and allele specific amplification). Oligonucleotidesnecessary for amplification may be selected for example, from within theIL-1 gene loci, either flanking the marker of interest (as required forPCR amplification) or directly overlapping the marker (as in ASOhybridization). In a particularly preferred embodiment, the sample ishybridized with a set of primers, which hybridize 5′ and 3′ in a senseor antisense sequence to the vascular disease associated allele, and issubjected to a PCR amplification.

[0036] An allele associated with a cardiovascular disorder may also bedetected indirectly, e.g. by analyzing the protein product encoded bythe DNA. For example, where the marker in question results in thetranslation of a mutant protein, the protein can be detected by any of avariety of protein detection methods. Such methods includeimmunodetection and biochemical tests, such as size fractionation, wherethe protein has a change in apparent molecular weight either throughtruncation, elongation, altered folding or altered post-translationalmodifications.

[0037] In another aspect, the invention features kits for performing theabove-described assays. The kit can include a nucleic acid samplecollection means and a means for determining whether a subject carries acardiovascular disorder associated allele. The kit may also contain acontrol sample either positive or negative or a standard and/or analgorithmic device for assessing the results and additional reagents andcomponents including: DNA amplification reagents, DNA polymerase,nucleic acid amplification reagents, restrictive enzymes, buffers, anucleic acid sampling device, DNA purification device, deoxynucleotides,oligonucleotides (e.g. probes and primers) etc.

[0038] As described above, the control samples may be positive ornegative controls. Further, the control sample may contain the positive(or negative) products of the allele detection technique employed. Forexample, where the allele detection technique is PCR amplification,followed by size fractionation, the control sample may comprise DNAfragments of the appropriate size. Likewise, where the allele detectiontechnique involves detection of a mutated protein, the control samplemay comprise a sample of mutated protein. However, it is preferred thatthe control sample comprises the material to be tested. For example, thecontrols may be a sample of genomic DNA or a cloned portion of the IL-1gene cluster. Preferably, however, the control sample is a highlypurified sample of genomic DNA where the sample to be tested is genomicDNA.

[0039] The oligonucleotides present in said kit may be used foramplification of the region of interest or for direct allele specificoligonucleotide (ASO) hybridization to the markers in question. Thus,the oligonucleotides may either flank the marker of interest (asrequired for PCR amplification) or directly overlap the marker (as inASO hybridization).

[0040] Information obtained using the assays and kits described herein(alone or in conjunction with information on a risk factor, such as aconcurrent disease, a genetic defect or environmental factor whichcontributes to a vascular disorder) is useful for determining whether anon-symptomatic subject has a cardiovascular disorder or is likely todevelop a cardiovascular disease. In addition, the information can allowa more customized approach and allow one to determine whether the courseof action should involve the use of more invasive procedures or whethertreatment should be aimed at plaque stabilization. This information canenable a clinician to more effectively prescribe a therapy that willaddress the molecular or genetic basis of the disorder.

[0041] In a further aspect, the invention features methods for treatingor preventing the development of a cardiovascular disorder in a subjectby administering to the subject an appropriate therapeutic of theinvention. In still another aspect, the invention provides in vitro orin vivo assays for screening test compounds to identify therapeutics fortreating or preventing a cardio-vascular disorder. In one embodiment,the assay comprises contacting a cell transfected with a causativemutation that is operably linked to an appropriate promoter with a testcompound and determining the level of expression of a protein in thecell in the presence and in the absence of the test compound. In oneembodiment, the causative mutation affects the systemic levels of IL-1receptor antagonist, and is associated with increased serum levels ofIL-1RA, so that the therapeutic efficacy of a particular compound can begauged by whether serum levels of IL-1RA fall in its presence. Inanother preferred embodiment, if the cardiovascular disorder causativemutation results in increased production of IL-1α or IL-1β, anddecreased production of IL-1α or IL-1β in the presence of the testcompound indicates that the compound is an antagonist of IL-1α or IL-1βactivity. In another embodiment, the invention features transgenicnon-human animals and their use in identifying antagonists of IL-1α orIL-1β activity or agonists of IL-1Ra activity.

[0042] Other features and advantages of the invention are set forth inthe following detailed description and claims.

BRIEF DESCRIPTION OF THE FIGURES

[0043]FIG. 1 depicts schematically a position of genes on Chromosome 2.

[0044]FIG. 2 shows a table of disequilibrium values within the IL-1 genecluster.

[0045]FIG. 3 presents a bar graph showing frequencies of haplotypepatterns.

[0046]FIG. 4 presents a schematic depiction of the alleles in IL-1Genotype Pattern 2 and certain of their clinical correlations.

[0047]FIG. 5 shows features of a clinical trial related to geneticmarkers.

[0048]FIG. 6 presents a bar graph of the association between an IL-1genotype and coronary artery stenosis.

[0049]FIG. 7 presents a bar graph showing associations between IL-1genotype patterns and restenosis.

[0050]FIG. 8 presents a bar graph showing associations betweenhomozygous and heterozygous allelic patterns at IL-1RN(+2018) locus andrestenosis and target vessel revascularization (TVR).

[0051]FIG. 9 presents a schematic flow chart of the relations betweencholesterol levels, IL-1 patterns and clinical events.

[0052]FIG. 10 presents a bar graph showing relationship between IL-1polymorphisms and risk for fragile plaque type clinical events withdifferent total cholesterol levels.

[0053]FIG. 11 presents a schematic depiction of the alleles in IL-1Genotype Pattern 1 and certain of their clinical correlations.

[0054]FIG. 12 shows a bar graph relating IL-1 genotype pattern 2 withLp(a) levels.

[0055]FIG. 13 shows a bar graph relating IL-1 genotype pattern 2 withLDL levels.

[0056]FIG. 14 shows a bar graph illustrating relationships between IL-1genotypes and levels of C-reactive protein.

DETAILED DESCRIPTION OF THE INVENTION

[0057] 4.1 Definitions

[0058] For convenience, the meaning of certain terms and phrasesemployed in the specification, examples, and appended claims areprovided below.

[0059] The term “aberrant activity”, as applied to an activity of apolypeptide such as IL-1, refers to an activity which differs from theactivity of a native polypeptide or which differs from the activity ofthe polypeptide in a healthy subject. An activity of a polypeptide canbe aberrant because it is stronger than the activity of its nativecounterpart. Alternatively, an activity can be aberrant because it isweaker or absent relative to the activity of its native counterpart. Anaberrant activity can also be a change in an activity. For example anaberrant polypeptide can interact with a different target peptide. Acell can have an aberrant IL-1 activity due to overexpression orunderexpression of an IL-1 locus gene encoding an IL-1 locuspolypeptide.

[0060] The term “allele” refers to the different sequence variants foundat different polymorphic regions. For example, IL-1RN (VNTR) has atleast five different alleles. The sequence variants may be single ormultiple base changes, including without limitation insertions,deletions, or substitutions, or may be a variable number of sequencerepeats.

[0061] The term “allelic pattern” refers to the identity of an allele oralleles at one or more polymorphic regions. For example, an allelicpattern may consist of a single allele at a polymorphic site, as forIL-1RN (VNTR) allele 1, which is an allelic pattern having at least onecopy of IL-1RN allele 1 at the VNTR of the IL-1RN gene loci.Alternatively, an allelic pattern may consist of either a homozygous orheterozygous state at a single polymorphic site. For example, IL1-RN(VNTR) allele 2,2 is an allelic pattern in which there are two copies ofthe second allele at the VNTR marker of IL-1RN and that corresponds tothe homozygous IL-RN (VNTR) allele 2 state. Alternatively, an allelicpattern may consist of the identity of alleles at more than onepolymorphic site.

[0062] The term “antibody” as used herein is intended to refer to abinding agent including a whole antibody or a binding fragment thereofwhich is specifically reactive with an IL-1B polypeptide. Antibodies canbe fragmented using conventional techniques and the fragments screenedfor utility in the same manner as described above for whole antibodies.For example, F(ab)₂ fragments can be generated by treating an antibodywith pepsin. The resulting F(ab)₂ fragment can be treated to reducedisulfide bridges to produce Fab fragments. The antibody of the presentinvention is further intended to include bispecific, single-chain, andchimeric and humanized molecules having affinity for an IL-1Bpolypeptide conferred by at least one CDR region of the antibody.

[0063] “Biological activity” or “bioactivity” or “activity” or“biological function”, which are used interchangeably, for the purposesherein when applied to IL-1 means an effector or antigenic function thatis directly or indirectly performed by an IL-1 polypeptide (whether inits native or denatured conformation), or by any subsequence (fragment)thereof A biological activity can include binding, effecting signaltransduction from a receptor, modulation of gene expression or anantigenic effector function.

[0064] As used herein the term “bioactive fragment of an IL-1polypeptide” refers to a fragment of a full-length IL-1 polypeptide,wherein the fragment specifically mimics or antagonizes the activity ofa wild-type IL-1 polypeptide. The bioactive fragment preferably is afragment capable of interacting with an interleukin receptor.

[0065] “Cells”, “host cells” or “recombinant host cells” are terms usedinterchangeably herein to refer not only to the particular subject cell,but to the progeny or potential progeny of such a cell. Because certainmodifications may occur in succeeding generations due to either mutationor environmental influences, such progeny may not, in fact be identicalto the parent cell, but is still included within the scope of the termas used herein.

[0066] A “chimera,” “mosaic,” “chimeric mammal” and the like, refers toa transgenic mammal with a knock-out or knock-in construct in at leastsome of its genome-containing cells.

[0067] The terms “control” or “control sample” refer to any sampleappropriate to the detection technique employed. The control sample maycontain the products of the allele detection technique employed or thematerial to be tested. Further, the controls may be positive or negativecontrols. By way of example, where the allele detection technique is PCRamplification, followed by size fractionation, the control sample maycomprise DNA fragments of an appropriate size. Likewise, where theallele detection technique involves detection of a mutated protein, thecontrol sample may comprise a sample of a mutant protein. However, it ispreferred that the control sample comprises the material to be tested.For example, the controls may be a sample of genomic DNA or a clonedportion of the IL-1 gene cluster. However, where the sample to be testedis genomic DNA, the control sample is preferably a highly purifiedsample of genomic DNA.

[0068] A “cardiovascular disease” is a cardiovascular disorder, asdefined herein, characterized by clinical events including clinicalsymptoms and clinical signs. Clinical symptoms are those experiencesreported by a patient that indicate to the clinician the presence ofpathology. Clinical signs are those objective findings on physical orlaboratory examination that indicate to the clinician the presence ofpathology. “Cardiovascular disease” includes both “coronary arterydisease” and “peripheral vascular disease,” both terms being definedbelow. Clinical symptoms in cardiovascular disease include chest pain,shortness of breath, weakness, fainting spells, alterations inconsciousness, extremity pain, paroxysmal nocturnal dyspnea, transientischemic attacks and other such phenomena experienced by the patient.Clinical signs in cardiovascular disease include such findings as EKGabnormalities, altered peripheral pulses, arterial bruits, abnormalheart sounds, rales and wheezes, jugular venous distention, neurologicalalterations and other such findings discerned by the clinician. Clinicalsymptoms and clinical signs can combine in a cardiovascular disease suchas a myocardial infarction (MI) or a stroke (also termed a“cerebrovascular accident” or “CVA”), where the patient will reportcertain phenomena (symptoms) and the clinician will perceive otherphenomena (signs) all indicative of an underlying pathology.“Cardiovascular disease” includes those diseases related to thecardiovascular disorders of fragile plaque disorder, occlusive disorderand stenosis. For example, a cardiovascular disease resulting from afragile plaque disorder, as that term is defined below, can be termed a“fragile plaque disease.” Clinical events associated with fragile plaquedisease include those signs and symptoms where the rupture of a fragileplaque with subsequent acute thrombosis or with distal embolization arehallmarks. Examples of fragile plaque disease include certain strokesand myocardial infarctions. As another example, a cardiovascular diseaseresulting from an occlusive disorder can be termed an “occlusivedisease.” Clinical events associated with occlusive disease includethose signs and symptoms where the progressive occlusion of an arteryaffects the amount of circulation that reaches a target tissue.Progressive arterial occlusion may result in progressive ischemia thatmay ultimately progress to tissue death if the amount of circulation isinsufficient to maintain the tissues. Signs and symptoms of occlusivedisease include claudication, rest pain, angina, and gangrene, as wellas physical and laboratory findings indicative of vessel stenosis anddecreased distal perfusion. As yet another example, a cardiovasculardisease resulting from restenosis can be termed an in-stent stenosisdisease. In-stent stenosis disease includes the signs and symptomsresulting from the progressive blockage of an arterial stent that hasbeen positioned as part of a procedure like a percutaneous transluminalangioplasty, where the presence of the stent is intended to help holdthe vessel in its newly expanded configuration. The clinical events thataccompany in-stent stenosis disease are those attributable to therestenosis of the reconstructed artery.

[0069] A “cardiovascular disorder” refers broadly to both to coronaryartery disorders and peripheral arterial disorders. The term“cardiovascular disorder” can apply to any abnormality of an artery,whether structural, histological, biochemical or any other abnormality.This term includes those disorders characterized by fragile plaque(termed herein “fragile plaque disorders”), those disorderscharacterized by vaso-occlusion (termed herein “occlusive disorders”),and those disorders characterized by restenosis. A “cardiovasculardisorder” can occur in an artery primarily, that is, prior to anymedical or surgical intervention. Primary cardiovascular disordersinclude, among others, atherosclerosis, arterial occlusion, aneurysmformation and thrombosis. A “cardiovascular disorder” can occur in anartery secondarily, that is, following a medical or surgicalintervention. Secondary cardiovascular disorders include, among others,post-traumatic aneurysm formation, restenosis, and post-operative graftocclusion.

[0070] A “cardiovascular disorder causative functional mutation” refersto a mutation which causes or contributes to the development of acardiovascular disorder in a subject. Preferred mutations occur withinthe IL-1 complex. A cardiovascular disorder causative functionalmutation occurring within an IL-1 gene (e.g. IL-1A, IL-1B or IL-1RN) ora gene locus, which is linked thereto, may alter, for example, the openreading frame or splicing pattern of the gene, thereby resulting in theformation of an inactive or hypoactive gene product. For example, amutation which occurs in intron 6 of the IL-1A locus corresponds to avariable number of tandem repeat 46 bp sequences corresponding to fromfive to 18 repeat units (Bailly, et al. (1993) Eur. J. Immunol. 23:1240-45). These repeat sequences contain three potential binding sitesfor transcriptional factors: an SP1 site, a viral enhancer element, anda glucocorticoid-responsive element; therefore individuals carryingIL-1A intron 6 VNTR alleles with large numbers of repeat units may besubject to altered transcriptional regulation of the IL-1A gene andconsequent perturbations of inflammatory cytokine production. Indeed,there is evidence that increased repeat number at this polymorphic IL-1Alocus leads to decreased IL-1α synthesis (Bailly et al. (1996) MolImmunol 33: 999-1006). Alternatively, a mutation can result in ahyperactive gene product. For example, allele 2 of the IL-1B (G at+6912) polymorphism occurs in the 3′ UTR (untranslated region) of theIL-1B mRNA and is associated with an approximately four-fold increase inthe steady state levels of both IL-1B mRNA and IL-1B protein compared tothose levels associated with allele 1 of the IL-1B gene at +6912).Further, an IL-1B (−511) mutation occurs near a promoter binding sitefor a negative glucocorticoid response element (Zhang et al. (1997) DNACell Biol 16: 145-52). This element potentiates a four-fold repressionof IL-1B expression by dexamethosone and a deletion of this negativeresponse elements causes a 2.5-fold increase in IL-1B promoter activity.The IL-1B (−511) polymorphism may thus directly affect cytokineproduction and inflammatory responses. These examples demonstrate thatgenetic variants occurring in the IL-1A or IL-1B gene can directly leadto the altered production or regulation of IL-1 cytokine activity.

[0071] A “cardiovascular disorder therapeutic” refers to any agent ortherapeutic regimen (including pharmaceuticals, nutraceuticals andsurgical means) that prevents or postpones the development of or reducesthe extent of an abnormality constitutive of a cardiovascular disorderin a subject. Cardiovascular disorder therapeutics can be directed tothe treatment of any cardiovascular disorder, including fragile plaquedisorder, occlusive disorder and restenosis. Examples of therapeuticagents directed to each category of cardiovascular disorder are providedherein. It is understood that a therapeutic agent may be useful for morethan one category of cardiovascular disorder. The therapeutic can be apolypeptide, peptidomimetic, nucleic acid or other inorganic or organicmolecule, preferably a “small molecule” including vitamins, minerals andother nutrients. Preferably the therapeutic can modulate at least oneactivity of an IL-1 polypeptide, e.g., interaction with a receptor, bymimicking or potentiating (agonizing) or inhibiting (antagonizing) theeffects of a naturally-occurring polypeptide. An IL-1 agonist can be awild-type protein or derivative thereof having at least one bioactivityof the wild-type, e.g., receptor binding activity. An IL-1 agonist canalso be a compound that upregulates expression of a gene or whichincreases at least one bioactivity of a protein. An IL-1 agonist canalso be a compound which increases the interaction of a polypeptide withanother molecule, e.g., a receptor. An IL-1 antagonist can be a compoundwhich inhibits or decreases the interaction between a protein andanother molecule, e.g., a receptor or an agent that blocks signaltransduction or post-translation processing (e.g., IL-1 convertingenzyme (ICE) inhibitor). Accordingly, a preferred antagonist is acompound which inhibits or decreases binding to a receptor and therebyblocks subsequent activation of the receptor. An IL-1 antagonist canalso be a compound that downregulates expression of a gene or whichreduces the amount of a protein present. The antagonist can be adominant negative form of a polypeptide, e.g., a form of a polypeptidewhich is capable of interacting with a target peptide, e.g., a receptor,but which does not promote the activation of the receptor. Theantagonist can also be a nucleic acid encoding a dominant negative formof a polypeptide, an antisense nucleic acid, or a ribozyme capable ofinteracting specifically with an RNA. Yet other antagonists aremolecules which bind to a polypeptide and inhibit its action. Suchmolecules include peptides, e.g., forms of target peptides which do nothave biological activity, and which inhibit binding to receptors. Thus,such peptides will bind to the active site of a protein and prevent itfrom interacting with target peptides. Yet other antagonists includeantibodies that specifically interact with an epitope of a molecule,such that binding interferes with the biological function of thepolypeptide. In yet another preferred embodiment, the antagonist is asmall molecule, such as a molecule capable of inhibiting the interactionbetween a polypeptide and a target receptor. Alternatively, the smallmolecule can function as an antagonist by interacting with sites otherthan the receptor binding site. Preferred therapeutics include lipidlowering drugs, antiplatelet agents, anti-inflammatory agents andantihypertensive agents.

[0072] “Cerebrovascular disease,” as used herein, is a type ofperipheral vascular disease (as defined below) where the peripheralvessel blocked is part of the cerebral circulation. The cerebralcirculation includes the carotid and the vertebral arterial systems.This definition of cerebrovascular disease is intended specifically toinclude intracranial hemorrhage that does not occur as a manifestationof an arterial blockage. Blockage can occur suddenly, by mechanisms suchas plaque rupture or embolization. Blockage can occur progressively,with narrowing of the artery via myointimal hyperplasia and plaqueformation. Blockage can be complete or partial. Certain degrees anddurations of blockage result in cerebral ischemia, a reduction of bloodflow that lasts for several seconds to minutes. The prolongation ofcerebral ischemia can result in cerebral infarction. Ischemia andinfarction can be focal or widespread. Cerebral ischemia or infarctioncan result in the abrupt onset of a non-convulsive focal neurologicaldefect, a clinical event termed a “stroke” or a “cerebrovascularaccident (CVA)”. Cerebrovascular disease has two broad categories ofpathologies: thrombosis and embolism. Thrombotic strokes occur withoutwarning symptoms in 80-90% of patients; between 10 and 20% of thromboticstrokes are heralded by transient ischemic attacks. A cerebrovasculardisease can be associated with a fragile plaque disorder. The signs andsymptoms of this type of cerebrovascular disease are those associatedwith fragile plaque, including stroke due to sudden arterial blockagewith thrombus or embolus formation. A cerebrovascular disease can beassociated with occlusive disorder. The signs and symptoms of this typeof cerebrovascular disease relate to progressive blockage of blood flowwith global or local cerebral ischemia. In this setting, neurologicalchanges can be seen, including stroke.

[0073] A “clinical event” is an occurrence of clinically discerniblesigns of a disease or of clinically reportable symptoms of a disease.“Clinically discernible” indicates that the sign can be appreciated by ahealth care provider. “Clinically reportable” indicates that the symptomis the type of phenomenon that can be described to a health careprovider. A clinical event may comprise clinically reportable symptomseven if the particular patient cannot himself or herself report them, aslong as these are the types of phenomena that are generally capable ofdescription by a patient to a health care provider.

[0074] A “coronary artery disease” (“CAD”) refers to a vascular disorderrelating to the blockage of arteries serving the heart. Blockage canoccur suddenly, by mechanisms such as plaque rupture or embolization.Blockage can occur progressively, with narrowing of the artery viamyointimal hyperplasia and plaque formation. Those clinical signs andsymptoms resulting from the blockage of arteries serving the heart aremanifestations of coronary artery disease. Manifestations of coronaryartery disease include angina, ischemia, myocardial infarction,cardiomyopathy, congestive heart failure, arrhythmias and aneurysmformation. It is understood that fragile plaque disease in the coronarycirculation is associated with arterial thrombosis or distalembolization that manifests itself as a myocardial infarction. It isunderstood that occlusive disease in the coronary circulation isassociated with arterial stenosis accompanied by anginal symptoms, acondition commonly treated with pharmacological interventions and withangioplasty.

[0075] A “disease” is a disorder characterized by clinical eventsincluding clinical signs and clinical symptoms. The diseases discussedherein include cardiovascular disease, peripheral vascular disease, CAD,cerebrovascular disease, and those diseases in any anatomic locationassociated with fragile plaque disorder, with occlusive disorder or withrestenosis.

[0076] A “disorder associated allele” or “an allele associated with adisorder” refers to an allele whose presence in a subject indicates thatthe subject has or is susceptible to developing a particular disorder.One type of disorder associated allele is a “cardiovascular disorderassociated allele,” the presence of which in a subject indicates thatthe subject has or is susceptible to developing a cardiovasculardisorder. These include broadly within their scope alleles which areassociated with “fragile plaque disorders,” alleles associated with“occlusive disorders,” and alleles associated with restenosis. Examplesof alleles associated with “fragile plaque disorders” include allele 2of the IL-1A +4825; allele 2 of the +3954 marker of IL-1B; and allele 1of the +2018 marker of IL-1RN; and allele 1 of the (−511 ) marker of theIL-1B gene or an allele that is in linkage disequilibrium with one ofthe aforementioned alleles. Examples of alleles associated with“occlusive disorders” include allele 1 of the IL-1A +4825; allele 1 ofthe +3954 marker of IL-1B; and allele 2 of the +2018 marker of IL-1RN;and allele 2 of the (−511) marker of the IL-1B gene or an allele that isin linkage disequilibrium with one of the aforementioned alleles.Examples of alleles associated with restenosis include the combinationof either allele 1 of the +4825 marker of IL-1A or allele 1 of the +3954marker as combined with either allele 1 of the −511 marker of IL-1B orallele 1 of the +2018 marker of IL-1RN, or an allele that is in linkagedisequilibrium with one of the aforementioned alleles. A “periodontaldisorder associated allele” refers to an allele whose presence in asubject indicates that the subject has or is susceptible to developing aperiodontal disorders.

[0077] The phrases “disruption of the gene” and “targeted disruption” orany similar phrase refers to the site specific interruption of a nativeDNA sequence so as to prevent expression of that gene in the cell ascompared to the wild-type copy of the gene. The interruption may becaused by deletions, insertions or modifications to the gene, or anycombination thereof.

[0078] As used herein, the terms “embolus,” “embolism” or “embolization”refer to artery-to-artery embolism or embolization.

[0079] “Fragile plaque disorder” refers to that cardiovascular disordercharacterized by the formation of fragile plaque as part of thearteriosclerotic process within an artery. The fragile plaque is proneto fracture, thrombosis or rupture. When the integrity of the plaque isaltered, it can mechanically block the vessel locally or it can sendfragments or associated clot downstream in the vessel to cause blockagemore distally. If the plaque cracks, it can be a nidus for a localthrombus to form. Fragile plaque disorder is associated with allelepattern 1 at the IL-1 locus.

[0080] A “fragile plaque disorder therapeutic” refers to any agent ortherapeutic regimen (including pharmaceuticals, nutraceuticals andsurgical means) that prevents or postpones the development of or reducesthe extent of an abnormality constitutive of a fragile plaque disorderin a subject. This term includes certain agents that operate bystabilizing fragile plaque, certain agents with anti-thrombotic oranti-platelet effect, and certain agents with antioxidant effect.Examples of fragile plaque disorder associated therapeutics includestatin drugs, anti-inflammatory agents with anti-prostaglandin effect,anti-inflammatory agents and cytokine inhibitors directed against IL-1and TNF-alpha such as Tenidap, matrix metalloproteanase (MMP) inhibitorsincluding tetracycline and related agents and specific MMP inhibitors,and recombinant IL-1 receptor antagonists. Furthermore, this termincludes those nutriceuticals that block IL-1, agents such as fish oils,omega-3 fatty acids, polyunsaturated fatty acids, and thosenutriceuticals with antioxidant effect such as butylated hydroxyanisol(BHA).

[0081] The term “haplotype” as used herein is intended to refer to a setof alleles that are inherited together as a group (are in linkagedisequilibrium) at statistically significant levels (p_(corr)<0.05). Asused herein, the phrase “an IL-1 haplotype” refers to a haplotype in theIL-1 loci.

[0082] An “IL-1 agonist” as used herein refers to an agent that mimics,upregulates (potentiates or supplements) or otherwise increases an IL-1bioactivity or a bioactivity of a gene in an IL-1 biological pathway.IL-1 agonists may act on any of a variety of different levels, includingregulation of IL-1 gene expression at the promoter region, regulation ofmRNA splicing mechanisms, stabilization of mRNA, phosphorylation ofproteins for translation, conversion of proIL-1 to mature IL-1 andsecretion of IL-1. Agonists that increase IL-1 synthesis include:lipopolysaccharides, IL-1B, cAMP inducing agents, NfκB activatingagents, AP-1 activating agents, TNF-α, oxidized LDL, advancedglycosylation end products (AGE), sheer stress, hypoxia, hyperoxia,ischemia reperfusion injury, histamine, prostaglandin E 2 (PGE2), IL-2,IL-3, IL-12, granulocyte macrophage-colony stimulating factor (GM-CSF),monocyte colony stimulating factor (M-CSF), stem cell factor, plateletderived growth factor (PDGF), complement C5A, complement C5b9, fibrindegradation products, plasmin, thrombin, 9-hydroxyoctadecaenoic acid,13-hydroxyoctadecaenoic acid, platelet activating factor (PAF), factorH, retinoic acid, uric acid, calcium pyrophosphate, polynucleosides,c-reactive protein, α-antitrypsin, tobacco antigen, collagen, β-1integrins, LFA-3, anti-HLA-DR, anti-IgM, anti-CD3, phytohemagglutinin(CD2), sCD23, ultraviolet B radiation, gamma radiation, substance P,.isoproterenol, methamphetamine and melatonin. Agonists that stabilizeIL-1 mRNA include bacterial endotoxin and IL-1. Other agonists, thatfunction by increasing the number of IL-1 type 1 receptors available,include IL-1, PKC activators, dexamethasone, IL-2, IL-4 and PGE2. Otherpreferred antagonists interfere or inhibit signal transduction factorsactivated by IL-1 or utilized in an IL-1 signal transduction pathway(e.g NFκB and AP-1, PI3 kinase, phospholipase A2, protein kinase C,JNK-1, 5-lipoxygenase, cyclooxygenase 2, tyrosine phosphorylation, iNOSpathway, Rac, Ras, TRAF). Still other agonists increase the bioactivityof genes whose expression is induced by IL-1, including: IL-1, IL-1Ra,TNF, IL-2, IL-3, IL-6, IL-12, GM-CSF, G-CSF, TGF-β, fibrinogen,urokinase plasminogen inhibitor, Type 1 and type 2 plasminogen activatorinhibitor, p-selectin (CD62), fibrinogen receptor, CD-11/CD18, proteasenexin-1, CD44, Matrix metalloproteinase-1 (MMP-1), MMP-3, Elastase,Collagenases, Tissue inhibitor of metalloproteinases-1 (TIMP-1),Collagen, Triglyceride increasing Apo CIII, Apolipoprotein, ICAM-1,ELAM-1, VCAM-1, L-selectin, Decorin, stem cell factor, Leukemiainhibiting factor, IFNα,β,γ, L-8, IL-2 receptor, IL-3 receptor, IL-5receptor, c-kit receptor, GM-CSF receptor, Cyclooxygenase-2 (COX-2),Type 2 phospholipase A2, Inducible nitric oxide synthase (iNOS),Endothelin-1,3, Gamma glutamyl transferase, Mn superoxide dismutase,C-reactive protein, Fibrinogen, Serum amyloid A, Metallothioneins,Ceruloplasmin, Lysozyme, Xanthine dehydrogenase, Xanthine oxidase,Platelet derived growth factor A chain (PDGF), Melanoma growthstimulatory activity (gro-α,β,γ), Insulin-like growth factor-1 (IGF-1),Activin A, Pro-opiomelanocortiotropin, corticotropin releasing factor, Bamyloid precursor, Basement membrane protein-40, Laminin B1 and B2,Constitutive heat shock protein p70, P42 mitogen, activating proteinkinase, ornithine decarboxylase, heme oxygenase and G-protein αsubunit).

[0083] An “IL-1 antagonist” as used herein refers to an agent thatdownregulates or otherwise decreases an IL-1 bioactivity. IL-1antagonists may act on any of a variety of different levels, includingregulation of IL-1 gene expression at the promoter region, regulation ofmRNA splicing mechanisms, stabilization of mRNA, phosphorylation ofproteins for translation, conversion of proIL-1 to mature IL-1 andsecretion of IL-1. Antagonists of IL-1 production include:corticosteroids, lipoxygenase inhibitors, cyclooxygenase inhibitors,γ-interferon, IL-4, IL-10, IL-13, transforming growth factor β (TGF-β),ACE inhibitors, n-3 polyunsaturated fatty acids, antioxidants and lipidreducing agents. Antagonists that destabilize IL-1mRNA include agentsthat promote deadenylation. Antagonists that inhibit or preventphosphorylation of IL-1 proteins for translation includepyridinyl-imadazole compounds, such as tebufelone and compounds thatinhibit microtubule formation (e.g. colchicine, vinblastine andvincristine). Antagonists that inhibit or prevent the conversion ofproIL-1 to mature IL-1 include interleukin converting enzyme (ICE)inhibitors, such as εICE isoforms, ICE α, β, and γ isoform antibodies,CXrm-A, transcript X, endogenous tetrapeptide competitive substrateinhibitor, trypsin, elastase, chymotrypsin, chymase, and othernonspecific proteases. Antagonists that prevent or inhibit the scretionof IL-1 include agents that block anion transport. Antagonists thatinterefere with IL-1 receptor interactions, include: agents that inhibitglycosylation of the type I IL-1 receptor, antisense oligonucleotidesagainst IL-1RI, antibodies to IL-1RI and antisense oligonucleotidesagainst IL-1RacP. Other antagonists, that function by decreasing thenumber of IL-1 type 1 receptors available, include TGF-β, COXinhibitors, factors that increase IL-1 type II receptors, dexamethasone,PGE2, IL-1 and IL-4. Other preferred antagonists interfere or inhibitsignal transduction factors activated by IL-1 or utilized in an IL-1signal transduction pathway (e.g NFκB and AP-1, PI3 kinase,phospholipase A2, protein kinase C, JNK-1, 5-lipoxygenase,cyclooxygenase 2, tyrosine phosphorylation, iNOS pathway, Rac, Ras,TRAF). Still other antagonists interfere with the bioactivity of geneswhose expression is induced by IL-1, including: IL-1, IL-1Ra, TNF, IL-2,IL-3, IL-6, IL-12, GM-CSF, G-CSF, TGF-β, fibrinogen, urokinaseplasminogen inhibitor, Type 1 and type 2 plasminogen activatorinhibitor, p-selectin (CD62), fibrinogen receptor, CD-11/CD18, proteasenexin-1, CD44, Matrix metalloproteinase-1 (MMP-1), MMP-3, Elastase,Collagenases, Tissue inhibitor of metalloproteinases-1 (TIMP-1),Collagen, Triglyceride increasing Apo CIII, Apolipoprotein, ICAM-1,ELAM-1, VCAM-1, L-selectin, Decorin, stem cell factor, Leukemiainhibiting factor, IFNα,β,γ, L-8, IL-2 receptor, IL-3 receptor, IL-5receptor, c-kit receptor, GM-CSF receptor, Cyclooxygenase-2 (COX-2),Type 2 phospholipase A2, Inducible nitric oxide synthase (iNOS),Endothelin-1,3, Gamma glutamyl transferase, Mn superoxide dismutase,C-reactive protein, Fibrinogen, Serum amyloid A, Metallothioneins,Ceruloplasmin, Lysozyme, Xanthine dehydrogenase, Xanthine oxidase,Platelet derived growth factor A chain (PDGF), Melanoma growthstimulatory activity (gro-α,β,γ), Insulin-like growth factor-1 (IGF-1),Activin A, Pro-opiomelanocortiotropin, corticotropin releasing factor, Bamyloid precursor, Basement membrane protein-40, Laminin B1 and B2,Constitutive heat shock protein p70, P42 mitogen, activating proteinkinase, ornithine decarboxylase, heme oxygenase and G-protein αsubunit). Other preferred antagonists include: hymenialdisine,herbimycines (e.g. herbamycin A), CK-103A and its derivatives (e.g.4,6-dihydropyridazino[4,5-c]pyridazin-5 (1H)-one), CK-119, CK-122,iodomethacin, aflatoxin B1, leptin, heparin, bicyclic imidazoles (e.gSB203580), PD15306 HCl, podocarpic acid derivatives, M-20, Human [Gly2]Glucagon-like peptide-2, FR167653, Steroid derivatives, glucocorticoids,Quercetin, Theophylline, NO-synthetase inhibitors, RWJ 68354, Euclyptol(1.8-cincole), Magnosalin, N-Acetylcysteine, Alpha-Melatonin-StimulatingHormone (α-MSH), Triclosan (2,4,4′-trichloro-2′-hydroxyldiphenyl ether),Prostaglandin E2 and 4-aminopyridine Ethacrynic acid and4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid (DIDS), Glucose,Lipophosphoglycan, aspirin, Catabolism-blocking agents, Diacerhein,Thiol-modulating agents, Zinc, Morphine, Leukotriene biosynthesisinhibitors (e.g. MK886), Platelet-activating factor receptor antagonists(e.g. WEB 2086), Amiodarone, Tranilast, S-methyl-L-thiocitrulline,Beta-adrenoreceptor agonists (e.g.Procaterol, Clenbuterol, Fenoterol,Terbutaline, Hyaluronic acid, anti-TNF-α antibodies, anti-IL-1αautoantibodies, IL-1 receptor antagonist, IL-1R-associated kinase,soluble TNF receptors and antiinflammatory cytokines (e.g IL-4, IL-13,IL-10, IL-6, TGF-β, angiotensin II, Soluble IL-1 type II receptor,Soluble IL-1 type I receptor, Tissue plasminogen activator, Zinc fingerprotein A20 IL-1 Peptides (e.g (Thr-Lys-Pro-Arg) (Tuftsin),(Ile-Thr-Gly-Ser-Glu) IL-1-alpha, Val-Thr-Lys-Phe-Tyr-Phe,Val-Thr-Asp-Phe-Tyr-Phe, Interferon alpha2b, Interferon beta, IL-1-betaanalogues (e.g. IL-1-beta tripeptide: Lys-D-Pro-Thr), glycosylatedIL-1-alpha, and IL-1ra peptides.

[0084] The terms “IL-1 gene cluster” and “IL-1 loci” as used hereininclude all the nucleic acid at or near the 2q13 region of chromosome 2,including at least the IL-1A, IL-1B and IL-1RN genes and any otherlinked sequences. (Nicklin et al., Genomics 19: 382-84, 1994). The terms“IL-1A”, “IL-1B”, and “IL-1RN” as used herein refer to the genes codingfor IL-1α, IL-1β, and IL-1 receptor antagonist, respectively. The geneaccession number for IL-1A, IL-1B, and IL-1RN are X03833, X04500, andX64532, respectively.

[0085] “IL-1 functional mutation” refers to a mutation within the IL-1gene cluster that results in an altered phenotype (i.e. affects thefunction of an IL-1 gene or protein). Examples include: IL-1A(+4845)allele 2, IL-1B (+3954) allele 2, IL-1B (−511) allele 1 and IL-1RN(+2018) allele 1.

[0086] “IL-1X (Z) allele Y” refers to a particular allelic form,designated Y, occurring at an IL-1 locus polymorphic site in gene X,wherein X is IL-1A, B, or RN or some other gene in the IL-1 gene loci,and positioned at or near nucleotide Z, wherein nucleotide Z is numberedrelative to the major transcriptional start site, which is nucleotide+1, of the particular IL-1 gene X. As further used herein, the term“IL-1X allele (Z)” refers to all alleles of an IL-1 polymorphic site ingene X positioned at or near nucleotide Z. For example, the term “IL-1RN(+2018) allele” refers to alternative forms of the IL-1RN gene at marker+2018. “IL-1RN (+2018) allele 1” refers to a form of the IL-1RN genewhich contains a cytosine (C) at position +2018 of the sense strand.Clay et al., Hum. Genet. 97:723-26, 1996. “IL-1RN (+2018) allele 2”refers to a form of the IL-1RN gene which contains a thymine (T) atposition +2018 of the plus strand. When a subject has two identicalIL-1RN alleles, the subject is said to be homozygous, or to have thehomozygous state. When a subject has two different IL-1RN alleles, thesubject is said to be heterozygous, or to have the heterozygous state.The term “IL-1RN (+2018) allele 2,2” refers to the homozygous IL-1 RN(+2018) allele 2 state. Conversely, the term “IL-1RN (+2018) allele 1,1”refers to the homozygous IL-1 RN (+2018) allele 1 state. The term“IL-1RN (+2018) allele 1,2” refers to the heterozygous allele 1 and 2state.

[0087] “IL-1 related” as used herein is meant to include all genesrelated to the human IL-1 locus genes on human chromosome 2 (2q 12-14).These include IL-1 genes of the human IL-1 gene cluster located atchromosome 2 (2q 13-14) which include: the IL-1A gene which encodesinterleukin-1α, the IL-1B gene which encodes interleukin-1β, and theIL-1RN (or IL-1ra) gene which encodes the interleukin-1 receptorantagonist. Furthermore these IL-1 related genes include the type I andtype II human IL-1 receptor genes located on human chromosome 2 (2q12)and their mouse homologs located on mouse chromosome 1 at position 19.5cM. Interleukin-1α, interleukin-1β, and interleukin-1RN are related inso much as they all bind to IL-1 type I receptors, however onlyinterleukin-1α and interleukin-1β are agonist ligands which activateIL-1 type I receptors, while interleukin-1RN is a naturally occurringantagonist ligand. Where the term “IL-1” is used in reference to a geneproduct or polypeptide, it is meant to refer to all gene productsencoded by the interleukin-1 locus on human chromosome 2 (2q 12-14) andtheir corresponding homologs from other species or functional variantsthereof. The term IL-1 thus includes secreted polypeptides which promotean inflammatory response, such as IL-1α and IL-1β, as well as a secretedpolypeptide which antagonize inflammatory responses, such as IL-1receptor antagonist and the IL-1 type II (decoy) receptor.

[0088] An “IL-1 receptor” or “IL-1R” refers to various cell membranebound protein receptors capable of binding to and/or transducing asignal from IL-1 locus-encoded ligand. The term applies to any of theproteins which are capable of binding interleukin-1 (IL-1) moleculesand, in their native configuration as mammalian plasma membraneproteins, presumably play a role in transducing the signal provided byIL-1 to a cell. As used herein, the term includes analogs of nativeproteins with IL-1-binding or signal transducing activity. Examplesinclude the human and murine IL-1 receptors described in U.S. Pat. No.4,968,607. The term “IL-1 nucleic acid” refers to a nucleic acidencoding an IL-1 protein.

[0089] An “IL-1 polypeptide” and “IL-1 protein” are intended toencompass polypeptides comprising the amino acid sequence encoded by theIL-1 genomic DNA sequences for IL-1α, IL-1β, and IL-1RN, or fragmentsthereof, and homologs thereof and include agonist and antagonistpolypeptides.

[0090] “In-stent stenosis” refers to the progressive occlusion within astent that has been placed during angioplasty. In-stent stenosis is aform of restenosis that takes place within an arterial stent.

[0091] “Increased risk” refers to a statistically higher frequency ofoccurrence of the disease or disorder in an individual in comparison tothe frequency of occurrence of the disease or disorder in a population.A factor identified to be associated with increased risk is termed a“risk factor.” Carrying a particular polymorphic allele is a risk factorfor a particular cardiovascular disease, and is associated with anincreased risk of the particular disease.

[0092] The term “interact” as used herein is meant to include detectablerelationships or associations (e.g. biochemical interactions) betweenmolecules, such as interactions between protein-protein, protein-nucleicacid, nucleic acid-nucleic acid and protein-small molecule or nucleicacid-small molecule in nature.

[0093] The term “isolated” as used herein with respect to nucleic acids,such as DNA or RNA, refers to molecules separated from other DNAs, orRNAs, respectively, that are present in the natural source of themacromolecule. For example, an isolated nucleic acid encoding one of thesubject IL-1 polypeptides preferably includes no more than 10 kilobases(kb) of nucleic acid sequence which naturally immediately flanks theIL-1 gene in genomic DNA, more preferably no more than 5 kb of suchnaturally occurring flanking sequences, and most preferably less than1.5 kb of such naturally occurring flanking sequence. The term isolatedas used herein also refers to a nucleic acid or peptide that issubstantially free of cellular material, viral material, or culturemedium when produced by recombinant DNA techniques, or chemicalprecursors or other chemicals when chemically synthesized. Moreover, an“isolated nucleic acid” is meant to include nucleic acid fragments whichare not naturally occurring as fragments and would not be found in thenatural state. The term “isolated” is also used herein to refer topolypeptides which are isolated from other cellular proteins and ismeant to encompass both purified and recombinant polypeptides.

[0094] A “knock-in” transgenic animal refers to an animal that has had amodified gene introduced into its genome and the modified gene can be ofexogenous or endogenous origin.

[0095] A “knock-out” transgenic animal refers to an animal in whichthere is partial or complete suppression of the expression of anendogenous gene (e.g, based on deletion of at least a portion of thegene, replacement of at least a portion of the gene with a secondsequence, introduction of stop codons, the mutation of bases encodingcritical amino acids, or the removal of an intron junction, etc.).

[0096] A “knock-out construct” refers to a nucleic acid sequence thatcan be used to decrease or suppress expression of a protein encoded byendogenous DNA sequences in a cell. In a simple example, the knock-outconstruct is comprised of a gene, such as the IL-1RN gene, with adeletion in a critical portion of the gene so that active protein cannotbe expressed therefrom. Alternatively, a number of termination codonscan be added to the native gene to cause early termination of theprotein or an intron junction can be inactivated. In a typical knock-outconstruct, some portion of the gene is replaced with a selectable marker(such as the neo gene) so that the gene can be represented as follows:IL-1RN5′/neo/IL-1RN3′, where IL-1RN5′ and IL-1RN 3′, refer to genomic orcDNA sequences which are, respectively, upstream and downstream relativeto a portion of the IL-1RN gene and where neo refers to a neomycinresistance gene. In another knock-out construct, a second selectablemarker is added in a flanking position so that the gene can berepresented as: IL-1RN/neo/IL-1RN/TK, where TK is a thymidine kinasegene which can be added to either the IL-1RN5′ or the IL-1RN3′ sequenceof the preceding construct and which further can be selected against(i.e. is a negative selectable marker) in appropriate media. Thistwo-marker construct allows the selection of homologous recombinationevents, which removes the flanking TK marker, from non-homologousrecombination events which typically retain the TK sequences. The genedeletion and/or replacement can be from the exons, introns, especiallyintron junctions, and/or the regulatory regions such as promoters.

[0097] “Linkage disequilibrium” refers to co-inheritance of two allelesat frequencies greater than would be expected from the separatefrequencies of occurrence of each allele in a given control population.The expected frequency of occurrence of two alleles that are inheritedindependently is the frequency of the first allele multiplied by thefrequency of the second allele. Alleles that co-occur at expectedfrequencies are said to be in “linkage disequilibrium”. The cause oflinkage disequilibrium is often unclear. It can be due to selection forcertain allele combinations or to recent admixture of geneticallyheterogeneous populations. In addition, in the case of markers that arevery tightly linked to a disease gene, an association of an allele (orgroup of linked alleles) with the disease gene is expected if thedisease mutation occurred in the recent past, so that sufficient timehas not elapsed for equilibrium to be achieved through recombinationevents in the specific chromosomal region. When referring to allelicpatterns that are comprised of more than one allele, a first allelicpattern is in linkage disequilibrium with a second allelic pattern ifall the alleles that comprise the first allelic pattern are in linkagedisequilibrium with at least one of the alleles of the second allelicpattern. An example of linkage disequilibrium is that which occursbetween the alleles at the IL-1RN (+2018) and IL-1RN (VNTR) polymorphicsites. The two alleles at IL-1RN (+2018) are 100% in linkagedisequilibrium with the two most frequent alleles of IL-1RN (VNTR),which are allele 1 and allele 2.

[0098] The term “marker” refers to a sequence in the genome that isknown to vary among individuals. For example, the IL-1RN gene has amarker that consists of a variable number of tandem repeats (VNTR).

[0099] “Modulate” refers to the ability of a substance to regulatebioactivity. When applied to an IL-1 bioactivity, an agonist orantagonist can modulate bioactivity for example by agonizing orantagonizing an IL-1 synthesis, receptor interaction, or IL-1 mediatedsignal transduction mechanism.

[0100] A “mutated gene” or “mutation” or “functional mutation” refers toan allelic form of a gene, which is capable of altering the phenotype ofa subject having the mutated gene relative to a subject which does nothave the mutated gene. The altered phenotype caused by a mutation can becorrected or compensated for by certain agents. If a subject must behomozygous for this mutation to have an altered phenotype, the mutationis said to be recessive. If one copy of the mutated gene is sufficientto alter the phenotype of the subject, the mutation is said to bedominant. If a subject has one copy of the mutated gene and has aphenotype that is intermediate between that of a homozygous and that ofa heterozygous subject (for that gene), the mutation is said to beco-dominant.

[0101] A “non-human animal” of the invention includes mammals such asrodents, non-human primates, sheep, dogs, cows, goats, etc. amphibians,such as members of the Xenopus genus, and transgenic avians (e.g.chickens, birds, etc.). The term “chimeric animal” is used herein torefer to animals in which the recombinant gene is found, or in which therecombinant gene is expressed in some but not all cells of the animal.The term “tissue-specific chimeric animal” indicates that one of therecombinant IL-1 genes is present and/or expressed or disrupted in sometissues but not others. The term “non-human mammal” refers to any memberof the class Mammalia, except for humans.

[0102] As used herein, the term “nucleic acid” refers to polynucleotidesor oligonucleotides such as deoxyribonucleic acid (DNA), and, whereappropriate, ribonucleic acid (RNA). The term should also be understoodto include, as equivalents, analogs of either RNA or DNA made fromnucleotide analogs (e.g. peptide nucleic acids) and as applicable to theembodiment being described, single (sense or antisense) anddouble-stranded polynucleotides.

[0103] “Occlusive disorder” refers to that cardiovascular disordercharacterized by the progressive thickening of an arterial wall,associated with the presence of an atherosclerotic intimal lesion withinan artery. Occlusive disorder leads to progressive blockage of theartery. With sufficient progression, the occlusive disorder can reduceflow in the artery to the point that clinical signs and symptoms areproduced in the tissues perfused by the artery. These clinical eventsrelate to ischemia of the perfused tissues. When severe, ischemia isaccompanied by tissue death, called infarction or gangrene. Occlusivedisorder is associated with the allele pattern 2s at the IL-1 locus.

[0104] An “occlusive disorder therapeutic” refers to any agent ortherapeutic regimen (including pharmaceuticals, nutraceuticals andsurgical means) that prevents or postpones the development of or reducesthe extent of an abnormality constitutive of an occlusive disorder in asubject. Examples of occlusive disorder therapeutics include thoseagents that are anti-oxidants, those that lower serum lipids, those thatblock the action of oxidized lipids and other agents that influencelipid metabolism or otherwise have lipid-active effects.

[0105] A “peripheral vascular disease” (“PVD”) is a cardiovasculardisease resulting from the blockage of the peripheral (i.e.,non-coronary) arteries. Blockage can occur suddenly, by mechanisms suchas plaque rupture or embolization, as occurs in fragile plaque disease.Blockage can occur progressively, with narrowing of the artery viamyointimal hyperplasia and plaque formation, as in occlusive disease.Blockage can be complete or partial. Those clinical signs and symptomsresulting from the blockage of peripheral arteries are manifestations ofperipheral vascular disease. Manifestations of peripheral vasculardiseases include, inter alia, claudication, ischemia, intestinal angina,vascular-based renal insufficiency, transient ischemic attacks, aneurysmformation, peripheral embolization and stroke. Ischemic cerebrovasculardisease is a type of peripheral vascular disease.

[0106] The term “polymorphism” refers to the coexistence of more thanone form of a gene or portion (e.g., allelic variant) thereof. A portionof a gene of which there are at least two different forms, i.e., twodifferent nucleotide sequences, is referred to as a “polymorphic regionof a gene”. A specific genetic sequence at a polymorphic region of agene is an allele. A polymorphic region can be a single nucleotide, theidentity of which differs in different alleles. A polymorphic region canalso be several nucleotides long.

[0107] The term “propensity to disease,” also “predisposition” or“susceptibility” to disease or any similar phrase, means that certainalleles are hereby discovered to be associated with or predictive of asubject's incidence of developing a particular disease (herein, acardiovascular disease). The alleles are thus over-represented infrequency in individuals with disease as compared to healthyindividuals. Thus, these alleles can be used to predict disease even inpre-symptomatic or pre-diseased individuals. These alleles areunderstood to relate to the disorder underlying the disease.

[0108] The term “restenosis” refers to any preocclusive lesion thatdevelops following a reconstructive procedure in a diseased bloodvessel. The term is not only applied to the recurrence of a pre-existingstenosis, but also to previously normal vessels such as vein grafts thatbecome partially occluded following vascular bypass. Restenosis refersto any luminal narrowing that occurs following a therapeuticintervention directed to an artery. Injuries resulting in restenosis cantherefore include trauma to an atherosclerotic lesion (as seen withangioplasty), a resection of a lesion (as seen with endarterectomy), anexternal trauma (e.g., a cross-clamping injury), or a surgicalanastomosis. Restenosis can occur as the result of any time of vascularreconstruction, whether in the coronary vasculature or in the periphery(Colburn and Moore (1998) Myointimal Hyperplasia pp. 690-709 in VascularSurgery: A Comprehensive Review (Philadelphia: Saunders, 1998)). Forexample, studies have reported symptomatic restenosis rates of 30-50%following coronary angioplasties (see Berk and Harris (1995) Adv.Intern. Med. 40:455-501). After carotid endarterectomies, as a furtherexample, 20% of patients studied had a luminal narrowing greater than50% (Clagett et al. (1986) J. Vasc. Surg. 3:10-23). Yet another exampleof restenosis is seen in infrainguinal vascular bypasses, where 40-60%of prosthetic grafts and 20-40% of the vein grafts are occluded at threeyears (Dalman and Taylor (1990) Ann. Vasc. Surg. 3:109-312, Szilagyi etal. (1973) Ann. Surg. 178:232-246). Different degrees of symptomatologyaccompany preocclusive lesions in different anatomical locations, due toa combination of factors including the different calibers of the vesselsinvolved, the extent of residual disease and local hemodynamics.In-stent stenosis is a type of restenosis.

[0109] A “restenosis disorder therapeutic” refers to any agent ortherapeutic regimen (including pharmaceuticals, nutraceuticals andsurgical means) that prevents or postpones the development of or reducesthe extent of an abnormality constitutive of a restenosis disorder in apatient. Restenosis is understood to comprise three phases. The firstphase is characterized by an inflammatory response involving therecruitment of leukocytes to the site of injury and by the formation ofthrombus during the first forty-eight hours. The endothelium isactivated with the expression of adhesion molecules ICAM-1, E-selectin,P-selectin and VCAM-1. At the same time, macrophages and fibroblastsbegin to migrate into the injury site by means of upregulation ofintegrins. The second phase is characterized by the proliferation ofsmooth muscle cells in the vessel wall media and the migration of thesecells into the intima where they migrate. Growth factors and cytokinesthat regulate the proliferation and migration of smooth muscle cells arereleased from the platelets, leukocytes and smooth muscle cells. Thelast phase includes the secretory phase of extracellular matrix fromsmooth muscle cells. A restenosis disorder therapeutics may act toaffect any of these processes in modulating the course of restenosis.Restenosis disorder therapeutics may include those agents that influencethe processes of NO synthesis, such as troglitazone and tranilast.Restenosis disorder therapeutics include physical interventions such asradiation therapies that influence the progression of restenosis or ofin-stent restenosis. Restenosis disorder therapeutics include stentmodification techniques such as seeding stents with genetically modifiedendothelial cells, coating stents with heparin or related agents,providing drug-loaded polymer stents, sconstructing polymer-coatedstents eluting platelet glycoprotein receptor antibodies or other stentmodifications. Restenosis disorder therapeutics include geneticengineering techniques, for example, those that involve transfer oftherapeutic genes or and those that involve incorporation of plasmid DNAin hydrogel coated medical devices. Restenosis disorder therapeuticsalso include surgical manipulations or parts of surgical treatment plansintended to minimize the incidence of restenosis or to avoid it.Restenosis disorders are understood to occur in all native arteriessubjected to endovascular manipulation and in autogenous veins used asvascular grafts. Intimal hyperplasia of either the proximal or thedistal anastomosis or of the vein graft itself continues to be theleading cause of late failures of infrainguinal vascularreconstructions, for example. In prosthetic graft reconstructions, suchproblems are extremely unusual. Diagnosing a propensity for an occlusivedisorder might guide the surgeon in selecting the type of graft materialto be used for a vascular reconstruction, or might influence the choiceof pharmacological agents to be used as adjuncts to the procedure.

[0110] A “risk factor” is a factor identified to be associated with anincreased risk. A risk factor for a cardiovascular disorder or acardiovascular disease is any factor identified to be associated with anincreased risk of developing those conditions or of worsening thoseconditions. A risk factor can also be associated with an increased riskof an adverse clinical event or an adverse clinical outcome in a patientwith a cardiovascular disorder. Risk factors for cardiovascular diseaseinclude smoking, adverse lipid profiles, elevated lipids or cholesterol,diabetes, hypertension, hypercoagulable states, elevated homocysteinelevels, and lack of exercise. Carrying a particular polymorphic alleleis a risk factor for a particular cardiovascular disorder, and isassociated with an increased risk of the particular disorder.

[0111] “Small molecule” as used herein, is meant to refer to acomposition, which has a molecular weight of less than about 5 kD andmost preferably less than about 4 kD. Small molecules can be nucleicacids, peptides, peptidomimetics, carbohydrates, lipids or other organicor inorganic molecules.

[0112] As used herein, the term “specifically hybridizes” or“specifically detects” refers to the ability of a nucleic acid moleculeto hybridize to at least approximately 6 consecutive nucleotides of asample nucleic acid.

[0113] “Stenosis,” as understood herein refers to a narrowing of anartery as seen in occlusive disorder or in restenosis. Stenosis can beaccompanied by those symptoms reflecting a decrease in blood flow pastthe narrowed arterial segment, in which case the disorder giving rise tothe stenosis is termed a disease (i.e., occlusive disease or restenosisdisease). Stenosis can exist asymptomatically in a vessel, to bedetected only by a diagnostic intervention such as an angiography or avascular lab study.

[0114] “Transcriptional regulatory sequence” is a generic term usedthroughout the specification to refer to DNA sequences, such asinitiation signals, enhancers, and promoters, which induce or controltranscription of protein coding sequences with which they are operablylinked.

[0115] As used herein, the term “transgene” means a nucleic acidsequence (encoding, e.g., one of the IL-1 polypeptides, or an antisensetranscript thereto) which has been introduced into a cell. A transgenecould be partly or entirely heterologous, i.e., foreign, to thetransgenic animal or cell into which it is introduced, or, is homologousto an endogenous gene of the transgenic animal or cell into which it isintroduced, but which is designed to be inserted, or is inserted, intothe animal's genome in such a way as to alter the genome of the cellinto which it is inserted (e.g., it is inserted at a location whichdiffers from that of the natural gene or its insertion results in aknockout). A transgene can also be present in a cell in the form of anepisome. A transgene can include one or more transcriptional regulatorysequences and any other nucleic acid, such as introns, that may benecessary for optimal expression of a selected nucleic acid.

[0116] A “transgenic animal” refers to any animal, preferably anon-human mammal, bird or an amphibian, in which one or more of thecells of the animal contain heterologous nucleic acid introduced by wayof human intervention, such as by transgenic techniques well known inthe art. The nucleic acid is introduced into the cell, directly orindirectly by introduction into a precursor of the cell, by way ofdeliberate genetic manipulation, such as by microinjection or byinfection with a recombinant virus. The term genetic manipulation doesnot include classical cross-breeding, or in vitro fertilization, butrather is directed to the introduction of a recombinant DNA molecule.This molecule may be integrated within a chromosome, or it may beextrachromosomally replicating DNA. In the typical transgenic animalsdescribed herein, the transgene causes cells to express a recombinantform of one of an IL-1 polypeptide, e.g. either agonistic orantagonistic forms. However, transgenic animals in which the recombinantgene is silent are also contemplated, as for example, the FLP or CRErecombinase dependent constructs described below. Moreover, “transgenicanimal” also includes those recombinant animals in which gene disruptionof one or more genes is caused by human intervention, including bothrecombination and antisense techniques. The term is intended to includeall progeny generations. Thus, the founder animal and all F1, F2, F3,and so on, progeny thereof are included.

[0117] The term “treating” as used herein is intended to encompasscuring as well as ameliorating at least one symptom of a disease or atleast one abnormality associated with a disorder. Treating acardiovascular disorder can take place by administering a cardiovasculardisorder therapeutic. Treating a cardiovascular disorder can also takeplace by modifying risk factors that are related to the cardiovasculardisorder.

[0118] A “treatment plan” refers to at least one intervention undertakento modify the effect of a risk factor upon a patient. A treatment planfor a cardiovascular disorder or disease can address those risk factorsthat pertain to cardiovascular disorders or diseases. A treatment plancan include an intervention that focuses on changing patient behavior,such as stopping smoking. A treatment plan can include an interventionwhereby a therapeutic agent is administered to a patient. As examples,cholesterol levels can be lowered with proper medication, and diabetescan be controlled with insulin. Nicotine addiction can be treated bywithdrawal medications. A treatment plan can include an interventionthat is diagnostic. The presence of the risk factor of hypertension, forexample, can give rise to a diagnostic intervention whereby the etiologyof the hypertension is determined. After the reason for the hypertensionis identified, further treatments may be administered.

[0119] The term “vector” refers to a nucleic acid molecule, which iscapable of transporting another nucleic acid to which it has beenlinked. One type of preferred vector is an episome, i.e., a nucleic acidcapable of extra-chromosomal replication. Preferred vectors are thosecapable of autonomous replication and/or expression of nucleic acids towhich they are linked. Vectors capable of directing the expression ofgenes to which they are operatively linked are referred to herein as“expression vectors”. In general, expression vectors of utility inrecombinant DNA techniques are often in the form of “plasmids” whichrefer generally to circular double stranded DNA loops which, in theirvector form are not bound to the chromosome. In the presentspecification, “plasmid” and “vector” are used interchangeably as theplasmid is the most commonly used form of vector. However, the inventionis intended to include such other forms of expression vectors whichserve equivalent functions and which become known in the artsubsequently hereto.

[0120] The term “wild-type allele” refers to an allele of a gene which,when present in two copies in a subject results in a wild-typephenotype. There can be several different wild-type alleles of aspecific gene, since certain nucleotide changes in a gene may not affectthe phenotype of a subject having two copies of the gene with thenucleotide changes.

[0121] 4.2 General

[0122] The kits and methods of the present invention rely at least inpart upon the novel finding that there is an association of patterns ofalleles at four polymorphic loci in the IL-1 gene cluster withcardiovascular disorders. These patterns are referred to herein patterns1, 2 and 3. Pattern 1 comprises an allelic pattern including allele 2 ofIL-1A (+4845) or IL-1B (+3954) and allele 1 of IL-1B (−511) or IL-1RN(+2018), or an allele that is in linkage disequilibrium with one of theaforementioned allele. In a preferred embodiment, this allelic patternpermits the diagnosis of fragile plaque disorder. Pattern 2 comprises anallelic pattern including allele 2 of IL-1B (−511) or IL-1RN (+2018) andallele 1 of IL-1A (+4845) or IL-1B (+3954), or an allele that is inlinkage disequilibrium with one of the aforementioned alleles. In apreferred embodiment, this allelic pattern permits the diagnosis offragile plaque disorder. Pattern 3 comprises an allelic patternincluding allele 1 of IL-1A (+4845) or allele 1 of IL-1B (+3954), andallele 1 of IL-1B (−511) or allele 1 of IL-1RN (+2018), or an allelethat is in linkage disequilibrium with one of the aforementionedalleles. In a preferred embodiment, this allelic pattern permits thediagnosis of a restenosis disorder. In one aspect, the present inventionprovides novel methods and kits for determining whether a subject has acardiovascular disorder. In one aspect, the invention discloses a methodand a kit for determining whether a subject has a fragile plaquedisorder. In one aspect, the invention discloses a method and a kit fordetermining whether a subject has an occlusive disorder. In one aspect,the invention discloses a method and a kit for determining whether asubject has a restenosis disorder.

[0123] Genes for IL-1α, IL-1β and IL-1RN are located in a cluster onchromosome 2, as shown in FIG. 1. Certain genes at the IL-1 locus areunderstood to be in linkage disequilibrium, as shown in FIG. 2.Furthermore, as FIG. 3 illustrates, patterns of haplotypes can beidentified, and their frequencies in populations can be ascertained. Thethree haplotype patterns, patterns 1, 2 and 3, may be defined by fourpolymorphic loci in the IL-1 gene cluster as shown in Table 1. TABLE 1IL-1A IL-1B Haplotypes (+4845) (+3954) IL-1B (−511) IL-1RN (+2018)Pattern 1 Allele 2 Allele 2 Allele 1 Allele 1 Pattern 2 Allele 1 Allele1 Allele 2 Allele 2 Pattern 3 Allele 1 Allele 1 Allele 1 Allele 1

[0124] Haplotype pattern 1 is associated with fragile plaque disorders.Haplotype pattern 2 is associated with occlusive disorders. Haplotypepattern 3 is associated with restenosis disorders. As discussed above,because these alleles are in linkage disequilibrium with other alleles,the detection of such other linked alleles can also indicate that thesubject has or is predisposed to the development of a cardiovasculardisorder.

[0125] Atherosclerotic plaque that is prone to rupture (fragile plaque,as seen in fragile plaque disorders) has certain structural, cellular,and molecular features. Rupture of the fibrous cap overlaying avulnerable plaque is the most common cause of coronary thrombosis.Typically fragile plaque has a large lipid core and a thin fibrous capthat is often infiltrated by inflammatory cells. The nature of the lipidforming the core is also of significance; for instance, lipid in theform of cholesterol ester softens the plaque and crystalline cholesterolmay have the opposite effect. Furthermore, it is seen that aninflammatory cell infiltrate is a marker of plaque vulnerability.Several factors such as oxidized lipoproteins, infectious agents, orautoantigens, such as heat shock proteins may incite a chronicinflammatory response in an atherosclerotic plaque. Influx of activatedmacrophages and T lymphocytes into the plaque follows, with subsequentinflux of cytokines and matrix-degrading proteins, leading to theweakening of the connective tissue framework of the plaque. Matrixmettaloproteinases and certain cytokines are important factors in thepathogenesis of plaque vulnerability. Following the diagnosis of afragile plaque disorder, therapeutics can be devised to address featuresof the fragile plaque like the abovementioned.

[0126] In one embodiment, the present invention discloses theassociation between significant coronary artery stenosis, increasedcarotid artery wall intimal-medial thickness and the IL-1 genotypepattern 2. The presence of Pattern 2 can be measured to determine a riskfactor for the development of CAD. This pattern, and itspathophysiological correlates, is illustrated in FIG. 4. A clinicaltrial, whose findings are synopsized on FIG. 5, was conducted todetermine the association between a genetic marker and the presence ofsymptomatic coronary artery stenosis. The results of this clinical trialare depicted in FIG. 6, where about 75% of those patients homozygous forallele 2 at an IL-1RN locus were determined to have significant coronaryartery stenosis.

[0127] In one embodiment, the present invention discloses the relationbetween restenosis and the pattern 3 genotype at the IL-1 locus. Asshown in FIG. 7, a study shows that the pattern 3 genotype is associatedwith about a three-fold increase in the risk for restenosis, while therisk is 0.5 for the pattern 2 genotype and 1.0 for the pattern 1genotype. FIG. 8, depicting data from the same study, shows that about40% of those subjects homozygous for allele 1 at IL1RN(+2018) havesignificant restenosis, and 25% have required target vesselrevascularization.

[0128] It is understood that there may be a relation between the effectsof certain risk factors on a patient afflicted with one of theabovementioned cardiovascular disorders and the development of therelated disease, and there may be a relation between the effects of riskfactors and the progression of the related disease. Diagnosis of theunderlying haplotype pattern can guide the clinician in makingrecommendations or in designing interventions to decrease the impact ofthe risk factors on the particular disorder or disease.

[0129] For example, the IL-1 genotype pattern in a patient can berelated to the effect of total cholesterol levels on cardiovasculardisorders and diseases. The presence of a certain serum cholesterollevel in the presence of a certain IL-1 genotype pattern is associatedwith a statistically determinable risk for coronary occlusive diseaseand fragile plaque disease. These associations are illustratedschematically in FIG. 9. FIG. 10 summarizes some of the data supportingthe associations. The data indicate that the presence of Pattern 1, evenin the presence of low serum cholesterol, is a strong predictor of therisk for fragile plaque type events. Fragile plaque type events are alsoobserved in patients with Pattern 2, although there is a strongcorrelation with the serum cholesterol level. General associations ofthe Il-1 genotype pattern 2 are summarized schematically in FIG. 11.

[0130] Using the methods and kits of the present invention, the Il-1genotype pattern may be related to the Lp(a) level in a subject todetermine an odds ratio for occlusive CAD. It is understood thatcholesterol is transported in body fluids in the form of lipoproteinparticles. The protein component of these aggregates have specificcell-targeting capabilities. Each lipoprotein particle is classified bydensity and contains 1) a major species of lipids that is the core, and2) a specific apolipoprotein that is the shell of the particle. LDL, forexample, has a cholesterol core with an apolipoprotein B-100 shell.Lipoprotein (a) [Lp(a)] is a lipoprotein that contains LDL and a proteinchain that mimics plasminogen. Lp(a) appears to have atherogenic andprothrombotic effects that interfere with plasminogen and tPA binding tofibrin and stimulate plasminogen activator inhibitor (PAI) synthesis.Studies have shown a relationship between Lp(a) and coronary arterydisease (CAD). A determination of the Il-2 genotype pattern versus theconcentration of Lp(a) shows the relationship between symptomaticcoronary artery stenosis and Lp(a) levels in Pattern 2 patients, arelationship illustrated in FIG. 12. Those patients homozygous forIL-1B(−511) allele 2 have a greatly increased risk for symptomaticstenosis, despite low Lp(a) levels. FIG. 13 shows the relation betweenelevated LDL and the risk for coronary artery occlusion, indicating theinterrelation between Pattern 2 and elevated serum lipid levels.

[0131] The methods and kits of the present invention may be used torelate the level of C-reactive protein (CRP) to the IL-1 genotypepattern. Over 50% of those pattern 1 subjects who were homozygous forallele 2 at IL-1B(+3954) were found to have a CRP greater than 0.20,while those pattern 2 subjects homozygous for allele 1 at IL-1B(+3954)had a CRP greater than 0.20 only about 28% of the time. These data areillustrated in FIG. 14.

[0132] 4.3 Predictive Medicine

[0133] 4.3.1. Polymorphisms Associated with Cardio-Vascular Disorders

[0134] The present invention is based at least in part, on theidentification of alleles that are associated (to a statisticallysignificant extent) with the development of a cardiovascular disorder insubjects. Therefore, detection of these alleles, alone or in conjunctionwith another means in a subject indicate that the subject has or ispredisposed to the development of a cardiovascular disorder. Forexample, as shown in the following examples, IL-1 polymorphic alleleswhich are associated with a propensity for developing a coronary arterydisorder or other vascular disorders caused by vascular occlusioninclude allele 2 of IL-1B (−511), allele 2 of IL-1RN (VNTR), allele 2 ofIL-1RN (+2018), allele 1 of IL-1A (+4845) or allele 1 of IL-1B (+3954)or an allele that is in linkage disequilibrium with one of theaforementioned alleles.

[0135] The present invention also discloses IL-1 polymorphic alleleswhich are associated with a propensity or a greater risk forcardiovascular diseases caused due to the rupture of fragile plaques.These include allele 2 of IL-1A (+4845), allele 2 of IL-1B (+3954),allele 1 of IL-1B (−511), and allele 1 of IL-1RN (+2018) or an allelethat is in linkage disequilibrium with one of the aforementionedalleles. This pattern is also associated with an increased risk fordeveloping severe adult periodontitis.

[0136] For example, allele 2 of IL-1B (−511) and allele 2 of IL-1RN(VNTR) are in linkage disequilibrium with one another and with a numberof other IL-1 polymorphisms which define the IL-1 (44112332) haplotype(Cox, et al. (1998) Am. J. Hum. Genet. 62: 1180-88). Specifically, the44112332 haplotype comprises the following genotype: allele 4 of the222/223 marker of IL-1A allele 4 of the gz5/gz6 marker of IL-1A allele 1of the −889 marker of IL-1A allele 1 of the +3954 marker of IL-1B allele2 of the −511 marker of IL-1B allele 3 of the gaat.p33330 marker allele3 of the Y31 marker allele 2 of the VNTR marker of IL-1RN

[0137] Thus, in alternative embodiments of the present invention,genotyping analysis at the 222/223 marker of IL-1A, the gz5/gz6 markerof IL-1A, the −889 marker of IL-1A, the +3954 marker of IL-1B, thegaat.p33330 marker of the IL-1B/IL-1RN intergenic region, or the Y31marker of the IL-1B/IL-1RN intergenic region is determined, and thepresence of allele 4 of the 222/223 marker of IL-1A, allele 4 of thegz5/gz6 marker of IL-1A, allele 1 of the −889 marker of IL-1A, allele 1of the +3954 marker of IL-1B, allele 3 of the gaat.p33330 marker, orallele 3 of the Y31 marker is indicative of an increased likelihood ofdeveloping a cardiovascular disorder, particularly disorders caused byocclusion of the arteries. In certain embodiments, diagnosing apropensity for an occlusive disease can lead to modification oflifestyle factors associated with increased incidence of occlusiveclinical events, or can result in the introduction of therapeuticmodalities to reduce the risk of occlusive symptoms or signs. In otherembodiments, diagnosing a propensity for an occlusive disease can alertthe clinician to explanations for otherwise difficult-to-diagnosediseases such as intestinal angina, renovascular hypertension andothers, situations where arterial stenosis may be responsible for thesymptoms and signs.

[0138] In addition, allele 2 of the IL-1RN (+2018) polymorphism (Clay etal. (1996) Hum Genet 97: 723-26), also referred to as exon 2 (8006)(GenBank:X64532 at 8006) is known to be in linkage disequilibrium withallele 2 of the IL-1RN (VNTR) polymorphic locus, which in turn is a partof the 44112332 human haplotype. Thus, allele 2 of the IL-1RN (+2018)locus (i.e. C at +2018), is an allelic variant associated with the44112332 haplotype and therefore provides an alternative target forprognostic genotyping analysis to determine an individual's likelihoodof developing a vascular disorder. Similarly, three other polymorphismsin an IL-1RN alternative exon (Exon lic, which produces an intracellularform of the gene product) are also in linkage disequilibrium with allele2 of IL-1RN (VNTR) (Clay et al. (1996) Hum Genet 97: 723-26). Theseinclude: the IL-1RN exon lic (1812) polymorphism (GenBank:X77090 at1812); the IL-1RN exon lic (1868) polymorphism (GenBank:X77090 at 1868);and the IL-1RN exon lic (1887) polymorphism (GenBank:X77090 at 1887).Furthermore yet another polymorphism in the promoter for thealternatively spliced intracellular form of the gene, the Pic (1731)polymorphism (GenBank:X77090 at 1731), is also in linkage disequilibriumwith allele 2 of the IL-1RN (VNTR) polymorphic locus (Clay et al. (1996)Hum Genet 97: 723-26). The corresponding sequence alterations for eachof these IL-1RN polymorphic loci is shown below. Exon lic-1 Exon lic-2Exon lic-3 Pic Exon 2 (1812 of (1868 of (1887 of (1731 of (+2018 of GB:GB: GB: GB: Allele # IL-1RN) X77090) X77090 X77090) X77090) 1 T G A G G2 C A G C A

[0139] For each of these polymorphic loci, the allele 2 sequence varianthas been determined to be in linkage disequilibrium with allele 2 of theIL-1RN (VNTR) locus (Clay et al. (1996) Hum Genet 97: 723-26).

[0140] Similarly, the 33221461, which is associated with an increasedrisk for developing fragile plaque diseases comprises the followinggenotype: allele 3 of the 222/223 marker of IL-1A allele 3 of thegz5/gz6 marker of IL-1A allele 2 of the −889 marker of IL-1A allele 2 ofthe +3954 marker of IL-1B allele 1 of the −511 marker of IL-1B allele 4of the gaat.p33330 marker allele 6 of the Y31 marker allele 1 of +2018of IL-1RN allele 2 of +4845 of IL-1A allele 1 of the VNTR marker ofIL-1RN

[0141] In alternative embodiments of the invention, genotyping analysisat the −889 marker of IL-1A, the gaat.p33330 marker of the IL-1B/IL-1RNintergenic region, the Y31 marker of the IL-1B/IL-1 RN intergenic regionis determined, and the presence of allele 1 of the −899 marker of theIL-1A, allele 4 of the gaat.p3330 marker, or allele 6 of the Y31 markeris indicative of cardio-vascular disorders, particularly of an increasedrisk for fragile plaque disorders. These disorders are understood tolead to clinical events via thrombosis and embolization. Often theclinical event is unheralded by previous signs of ischemia. Chronicischemia, as disclosed herein, is associated with occlusivecardiovascular disease rather than with fragile plaque disease. Earlydetection for the propensity for a catastrophic clinical event would bea significant addition to the current diagnostic armamentarium. Afragile plaque clinical event in the cerebrovascular circulation cancause a stroke or CVA by blocking cerebral vessels and causing acuteischemia that can lead to irreversible brain. infarction. A fragileplaque clinical event in the myocardial circulation can cause amyocardial infarction by blocking coronary vessels and causing acuteischemia that can lead to irreversible myocardial damage. A fragileplaque clinical event in the non-cerebral peripheral vasculature canlead to sudden onset of ischemia leading to gangrene and tissue loss.Since these fragile plaque clinical events may ensue without priorwarning, the identification of the genotype associated with increasedrisk can lead to increased clinical monitoring in these at-risksubjects, with earlier and more extensive diagnostic or therapeuticinterventios. In another embodiment these are also indicative ofincreased risk for developing severe adult periodontitis. In yet anotherembodiment, the presence of severe adult periodontitis is indicative ofan increased risk for fragile plaque disease.

[0142] In addition to the allelic patterns described above, as describedherein, one of skill in the art can readily identify other alleles(including polymorphisms and mutations) that are in linkagedisequilibrium with an allele associated with a cardio-vasculardisorder. For example, a nucleic acid sample from a first group ofsubjects without a cardio vascular disorder can be collected, as well asDNA from a second group of subjects with a cardio vascular disorder. Thenucleic acid sample can then be compared to identify those alleles thatare over-represented in the second group as compared with the firstgroup, wherein such alleles are presumably associated with a cardiovascular disorder. Alternatively, alleles that are in linkagedisequilibrium with a cardiovascular disorder associated allele can beidentified, for example, by genotyping a large population and performingstatistical analysis to determine which alleles appear more commonlytogether than expected. Preferably the group is chosen to be comprisedof genetically related individuals. Genetically related individualsinclude individuals from the same race, the same ethnic group, or eventhe same family. As the degree of genetic relatedness between a controlgroup and a test group increases, so does the predictive value ofpolymorphic alleles which are ever more distantly linked to adisease-causing allele. This is because less evolutionary time haspassed to allow polymorphisms which are linked along a chromosome in afounder population to redistribute through genetic cross-over events.Thus race-specific, ethnic-specific, and even family-specific diagnosticgenotyping assays can be developed to allow for the detection of diseasealleles which arose at ever more recent times in human evolution, e.g.,after divergence of the major human races, after the separation of humanpopulations into distinct ethnic groups, and even within the recenthistory of a particular family line.

[0143] Linkage disequilibrium between two polymorphic markers or betweenone polymorphic marker and a disease-causing mutation is a meta-stablestate. Absent selective pressure or the sporadic linked reoccurrence ofthe underlying mutational events, the polymorphisms will eventuallybecome disassociated by chromosomal recombination events and willthereby reach linkage equilibrium through the course of human evolution.Thus, the likelihood of finding a polymorphic allele in linkagedisequilibrium with a disease or condition may increase with changes inat least two factors: decreasing physical distance between thepolymorphic marker and the disease-causing mutation, and decreasingnumber of meiotic generations available for the dissociation of thelinked pair. Consideration of the latter factor suggests that, the moreclosely related two individuals are, the more likely they will share acommon parental chromosome or chromosomal region containing the linkedpolymorphisms and the less likely that this linked pair will have becomeunlinked through meiotic cross-over events occurring each generation. Asa result, the more closely related two individuals are, the more likelyit is that widely spaced polymorphisms may be co-inherited. Thus, forindividuals related by common race, ethnicity or family, the reliabilityof ever more distantly spaced polymorphic loci can be relied upon as anindicator of inheritance of a linked disease-causing mutation.

[0144] The oligonucleotides present in one embodiment of a kit accordingto the present invention may be used for amplification of the region ofinterest or for direct allele specific oligonucleotide (ASO)hybridization to the markers in question. Thus, the oligonucleotides mayeither flank the marker of interest (as required for PCR amplification)or directly overlap the marker (as in ASO hybridization). Examples ofappropriate primers for use in the above described detection methods,include: 5′-CTCAGCAACACTCCTAT-3′, (SEQ ID NO. 1)5′-TCCTGGTCTGCAGGTAA-3′, (SEQ ID NO. 2)

[0145] which can be used to amplify and type the human IL-1RN (VNTR)polymorphic locus; (SEQ ID NO. 3) 5′-CTA TCT GAG GAA CAA CCA ACT AGTAGC-3′; (SEQ ID NO. 4) 5′-TAG GAC ATT GCA CCT AGG GTT TGT -3′;

[0146] which can be used to amplify and type the human IL-1RN (+2018)polymorphic locus; 5′TGGCATTGATCTGGTTCATC 3′; (SEQ ID No: 5)5′GTTTAGGAATCTTCCCACTT 3′; (SEQ ID No: 6)

[0147] which can be used to amplify and type the human IL-1B (−511)polymorphic locus.; (SEQ ID NO: 7) 5′CTC AGG TGT CCT CGA AGA AAT CAA A3′; (SEQ ID NO: 8) 5′GCT TTT TTG CTG TGA GTC CCG 3′

[0148] which can be used to amplify and type the human IL-1B (+3954)polymorphic locus; and (SEQ ID NO: 9) 5′ATG GTT TTA GAA ATC ATC AAG CCTAGG GCA 3′ (SEQ ID NO: 10) 3′AAT GAA AGG AGG GGA GGA TGA CAG AAA TGT 3′

[0149] which can be used to amplify and type the human IL-1A (+4845)polymorphic locus.

[0150] Appropriate probes may be designed to hybridize to a specificgene of the IL-1 locus, such as IL-1A, IL-1B or IL-1RN or a relatedgene. Alternatively, these probes may incorporate other regions of therelevant genomic locus, including intergenic sequences. Indeed the IL-1region of human chromosome 2 spans some 400,000 base pairs and, assumingan average of one single nucleotide polymorphism every 1,000 base pairs,includes some 400 SNPs loci alone. Yet other polymorphisms available foruse with the immediate invention are obtainable from various publicsources. For example, the human genome database collects intragenicSNPs, is searchable by sequence and currently contains approximately2,700 entries (http://hgbase.interactiva.de). Also available is a humanpolymorphism database maintained by the Massachusetts Institute ofTechnology (MIT SNP database(http://www.genome.wi.mit.edu/SNP/human/index.html)). From such sourcesSNPs as well as other human polymorphisms may be found.

[0151] For example, examination of the IL-1 region of the human genomein any one of these databases reveals that the IL-1 locus genes areflanked by a centromere proximal polymorphic marker designatedmicrosatellite marker AFM220ze3 at 127.4 cM (centiMorgans) (see GenBankAce. No. Z17008) and a distal polymorphic marker designatedmicrosatellite anchor marker AFM087xa1 at 127.9 cM (see GenBank Acc. No.Z16545). These human polymorphic loci are both CA dinucleotide repeatmicrosatellite polymorphisms, and, as such, show a high degree ofheterozygosity in human populations. For example, one allele ofAFM220ze3 generates a 211 bp PCR amplification product with a 5′ primerof the sequence TGTACCTAAGCCCACCCTTTAGAGC (SEQ ID No. 14) and a 3′primer of the sequence TGGCCTCCAGAAACCTCCAA (SEQ ID No. 15).Furthermore, one allele of AFM087xa1 generates a 177 bp PCRamplification product with a 5′ primer of the sequenceGCTGATATTCTGGTGGGAAA (SEQ ID No. 16) and a 3′ primer of the sequenceGGCAAGAGCAAAACTCTGTC (SEQ ID No. 17). Equivalent primers correspondingto unique sequences occurring 5′ and 3′ to these human chromosome 2 CAdinucleotide repeat polymorphisms will be apparent to one of skill inthe art. Reasonable equivalent primers include those which hybridizewithin about 1 kb of the designated primer, and which further areanywhere from about 17 bp to about 27 bp in length. A general guidelinefor designing primers for amplification of unique human chromosomalgenomic sequences is that they possess a melting temperature of at leastabout 50° C., wherein an approximate melting temperature can beestimated using the formula T_(melt)=[2×(# of A or T)+4×(# of G or C)].

[0152] A number of other human polymorphic loci occur between these twoCA dinucleotide repeat polymorphisms and provide additional targets fordetermination of a cardio-vascular disorder prognostic allele in afamily or other group of genetically related individuals. For example,the National Center for Biotechnology Information web site(www.ncbi.nlm.nih.gov/genemap/) lists a number of polymorphism markersin the region of the IL-1 locus and provides guidance in designingappropriate primers for amplification and analysis of these markers.

[0153] Accordingly, the nucleotide segments of the invention may be usedfor their ability to selectively form duplex molecules withcomplementary stretches of human chromosome 2 q 12-13 or cDNAs from thatregion or to provide primers for amplification of DNA or cDNA from thisregion. The design of appropriate probes for this purpose requiresconsideration of a number of factors. For example, fragments having alength of between 10, 15, or 18 nucleotides to about 20, or to about 30nucleotides, will find particular utility. Longer sequences, e.g., 40,50, 80, 90, 100, even up to full length, are even more preferred forcertain embodiments. Lengths of oligonucleotides of at least about 18 to20 nucleotides are well accepted by those of skill in the art assufficient to allow sufficiently specific hybridization so as to beuseful as a molecular probe. Furthermore, depending on the applicationenvisioned, one will desire to employ varying conditions ofhybridization to achieve varying degrees of selectivity of probe towardstarget sequence. For applications requiring high selectivity, one willtypically desire to employ relatively stringent conditions to form thehybrids. For example, relatively low salt and/or high temperatureconditions, such as provided by 0.02 M-0.15M NaCl at temperatures ofabout 50° C. to about 70° C. Such selective conditions may toleratelittle, if any, mismatch between the probe and the template or targetstrand.

[0154] Other alleles or other indicia of a vascular disorder can bedetected or monitored in a subject in conjunction with detection of thealleles described above, for example, identifying vessel wall thickness(e.g. as measured by ultrasound), or whether the subject smokes, drinks,is overweight, is under stress, has elevated cholesterol or lowcholesterol. has elevated Lp(a), or exercises.

[0155] 4.3.2 Detection of Alleles

[0156] Many methods are available for detecting specific alleles athuman polymorphic loci. The preferred method for detecting a specificpolymorphic allele will depend, in part, upon the molecular nature ofthe polymorphism. For example, the various allelic forms of thepolymorphic locus may differ by a single base-pair of the DNA. Suchsingle nucleotide polymorphisms (or SNPs) are major contributors togenetic variation, comprising some 80% of all known polymorphisms, andtheir density in the human genome is estimated to be on average 1 per1,000 base pairs. SNPs are most frequently biallelic-occurring in onlytwo different forms (although up to four different forms of an SNP,corresponding to the four different nucleotide bases occurring in DNA,are theoretically possible). Nevertheless, SNPs are mutationally morestable than other polymorphisms, making them suitable for associationstudies in which linkage disequilibrium between markers and an unknownvariant is used to map disease-causing mutations. In addition, becauseSNPs typically have only two alleles, they can be genotyped by a simpleplus/minus assay rather than a length measurement, making them moreamenable to automation.

[0157] A variety of methods are available for detecting the presence ofa particular single nucleotide polymorphic allele in an individual.Advancements in this field have provided accurate, easy, and inexpensivelarge-scale SNP genotyping. Most recently, for example, several newtechniques have been described including dynamic allele-specifichybridization (DASH), microplate array diagonal gel electrophoresis(MADGE), pyrosequencing, oligonucleotide-specific ligation, the TaqMansystem as well as various DNA “chip” technologies such as the AffymetrixSNP chips. These methods require amplification of the target geneticregion, typically by PCR. Still other newly developed methods, based onthe generation of small signal molecules by invasive cleavage followedby mass spectrometry or immobilized padlock probes and rolling-circleamplification, might eventually eliminate the need for PCR. Several ofthe methods known in the art for detecting specific single nucleotidepolymorphisms are summarized below. The method of the present inventionis understood to include all available methods.

[0158] Several methods have been developed to facilitate analysis ofsingle nucleotide polymorphisms. In one embodiment, the single basepolymorphism can be detected by using a specializedexonuclease-resistant nucleotide, as disclosed, e.g., in Mundy, C. R.(U.S. Pat. No. 4,656,127). According to the method, a primercomplementary to the allelic sequence immediately 3′ to the polymorphicsite is permitted to hybridize to a target molecule obtained from aparticular animal or human. If the polymorphic site on the targetmolecule contains a nucleotide that is complementary to the particularexonuclease-resistant nucleotide derivative present, then thatderivative will be incorporated onto the end of the hybridized primer.Such incorporation renders the primer resistant to exonuclease, andthereby permits its detection. Since the identity of theexonuclease-resistant derivative of the sample is known, a finding thatthe primer has become resistant to exonucleases reveals that thenucleotide present in the polymorphic site of the target molecule wascomplementary to that of the nucleotide derivative used in the reaction.This method has the advantage that it does not require the determinationof large amounts of extraneous sequence data.

[0159] In another embodiment of the invention, a solution-based methodis used for determining the identity of the nucleotide of a polymorphicsite. Cohen, D. et al. (French Patent 2,650,840; PCT Appln. No.WO91/02087). As in the Mundy method of U.S. Pat. No. 4,656,127, a primeris employed that is complementary to allelic sequences immediately 3′ toa polymorphic site. The method determines the identity of the nucleotideof that site using labeled dideoxynucleotide derivatives, which, ifcomplementary to the nucleotide of the polymorphic site will becomeincorporated onto the terminus of the primer.

[0160] An alternative method, known as Genetic Bit Analysis or GBA™ isdescribed by Goelet, P. et al. (PCT Appln. No. 92/15712). The method ofGoelet, P. et al. uses mixtures of labeled terminators and a primer thatis complementary to the sequence 3′ to a polymorphic site. The labeledterminator that is incorporated is thus determined by, and complementaryto, the nucleotide present in the polymorphic site of the targetmolecule being evaluated. In contrast to the method of Cohen et al.(French Patent 2,650,840; PCT Appln. No. WO91/02087) the method ofGoelet, P. et al. is preferably a heterogeneous phase assay, in whichthe primer or the target molecule is immobilized to a solid phase.

[0161] Recently, several primer-guided nucleotide incorporationprocedures for assaying polymorphic sites in DNA have been described(Komher, J. S. et al., Nucl. Acids. Res. 17:7779-7784 (1989); Sokolov,B. P., Nucl. Acids Res. 18:3671 (1990); Syvanen, A. -C., et al.,Genomics 8:684-692 (1990); Kuppuswamy, M. N. et al., Proc. Natl. Acad.Sci. (U.S.A.) 88:1143-1147 (1991); Prezant, T. R. et al., Hum. Mutat.1:159-164 (1992); Ugozzoli, L. et al., GATA 9:107-112 (1992); Nyren, P.et al., Anal. Biochem. 208:171-175 (1993)). These methods differ fromGBA™ in that they all rely on the incorporation of labeleddeoxynucleotides to discriminate between bases at a polymorphic site. Insuch a format, since the signal is proportional to the number ofdeoxynucleotides incorporated, polymorphisms that occur in runs of thesame nucleotide can result in signals that are proportional to thelength of the run (Syvanen, A. -C., et al., Amer. J. Hum. Genet.52:46-59 (1993)).

[0162] For mutations that produce premature termination of proteintranslation, the protein truncation test (PTT) offers an efficientdiagnostic approach (Roest, et. al., (1993) Hum. Mol. Genet. 2:1719-21;van der Luijt, et. al., (1994) Genomics 20:1-4). For PTT, RNA isinitially isolated from available tissue and reverse-transcribed, andthe segment of interest is amplified by PCR. The products of reversetranscription PCR are then used as a template for nested PCRamplification with a primer that contains an RNA polymerase promoter anda sequence for initiating eukaryotic translation. After amplification ofthe region of interest, the unique motifs incorporated into the primerpermit sequential in vitro transcription and translation of the PCRproducts. Upon sodium dodecyl sulfate-polyacrylamide gel electrophoresisof translation products, the appearance of truncated polypeptidessignals the presence of a mutation that causes premature termination oftranslation. In a variation of this technique, DNA (as opposed to RNA)is used as a PCR template when the target region of interest is derivedfrom a single exon.

[0163] Any cell type or tissue may be utilized to obtain nucleic acidsamples for use in the diagnostics described herein. In a preferredembodiment, the DNA sample is obtained from a bodily fluid, e.g, blood,obtained by known techniques (e.g. venipuncture) or saliva.Alternatively, nucleic acid tests can be performed on dry samples (e.g.hair or skin). When using RNA or protein, the cells or tissues that maybe utilized must express an IL-1 gene.

[0164] Diagnostic procedures may also be performed in situ directly upontissue sections (fixed and/or frozen) of patient tissue obtained frombiopsies or resections, such that no nucleic acid purification isnecessary. Nucleic acid reagents may be used as probes and/or primersfor such in situ procedures (see, for example, Nuovo, G. J., 1992, PCRin situ hybridization: protocols and applications, Raven Press, NY).

[0165] In addition to methods which focus primarily on the detection ofone nucleic acid sequence, profiles may also be assessed in suchdetection schemes. Fingerprint profiles may be generated, for example,by utilizing a differential display procedure, Northern analysis and/orRT-PCR.

[0166] A preferred detection method is allele specific hybridizationusing probes overlapping a region of at least one allele of an IL-1proinflammatory haplotype and having about 5, 10, 20, 25, or 30nucleotides around the mutation or polymorphic region. In a preferredembodiment of the invention, several probes capable of hybridizingspecifically to other allelic variants involved in a cardio-vasculardisorder are attached to a solid phase support, e.g., a “chip” (whichcan hold up to about 250,000 oligonucleotides). Oligonucleotides can bebound to a solid support by a variety of processes, includinglithography. Mutation detection analysis using these chips comprisingoligonucleotides, also termed “DNA probe arrays” is described e.g., inCronin et al. (1996) Human Mutation 7:244. In one embodiment, a chipcomprises all the allelic variants of at least one polymorphic region ofa gene. The solid phase support is then contacted with a test nucleicacid and hybridization to the specific probes is detected. Accordingly,the identity of numerous allelic variants of one or more genes can beidentified in a simple hybridization experiment.

[0167] These techniques may also comprise the step of amplifying thenucleic acid before analysis. Amplification techniques are known tothose of skill in the art and include, but are not limited to cloning,polymerase chain reaction (PCR), polymerase chain reaction of specificalleles (ASA), ligase chain reaction (LCR), nested polymerase chainreaction, self sustained sequence replication (Guatelli, J. C. et al.,1990, Proc. Natl. Acad. Sci. USA 87:1874-1878), transcriptionalamplification system (Kwoh, D. Y. et al., 1989, Proc. Natl. Acad. Sci.USA 86:1173-1177), and Q-Beta Replicase (Lizardi, P. M. et al., 1988,Bio/Technology 6:1197).

[0168] Amplification products may be assayed in a variety of ways,including size analysis, restriction digestion followed by sizeanalysis, detecting specific tagged oligonucleotide primers in thereaction products, allele-specific oligonucleotide (ASO) hybridization,allele specific 5′ exonuclease detection, sequencing, hybridization, andthe like.

[0169] PCR based detection means can include multiplex amplification ofa plurality of markers simultaneously. For example, it is well known inthe art to select PCR primers to generate PCR products that do notoverlap in size and can be analyzed simultaneously. Alternatively, it ispossible to amplify different markers with primers that aredifferentially labeled and thus can each be differentially detected. Ofcourse, hybridization based detection means allow the differentialdetection of multiple PCR products in a sample. Other techniques areknown in the art to allow multiplex analyses of a plurality of markers.

[0170] In a merely illustrative embodiment, the method includes thesteps of (i) collecting a sample of cells from a patient, (ii) isolatingnucleic acid (e.g., genomic, mRNA or both) from the cells of the sample,(iii) contacting the nucleic acid sample with one or more primers whichspecifically hybridize 5′ and 3′ to at least one allele of an IL-1proinflammatory haplotype under conditions such that hybridization andamplification of the allele occurs, and (iv) detecting the amplificationproduct. These detection schemes are especially useful for the detectionof nucleic acid molecules if such molecules are present in very lownumbers.

[0171] In a preferred embodiment of the subject assay, the allele of anIL-1 proinflammatory haplotype is identified by alterations inrestriction enzyme cleavage patterns. For example, sample and controlDNA is isolated, amplified (optionally), digested with one or morerestriction endonucleases, and fragment length sizes are determined bygel electrophoresis.

[0172] In yet another embodiment, any of a variety of sequencingreactions known in the art can be used to directly sequence the allele.Exemplary sequencing reactions include those based on techniquesdeveloped by Maxim and Gilbert ((1977) Proc. Natl Acad Sci USA 74:560)or Sanger (Sanger et al (1977) Proc. Nat. Acad. Sci USA 74:5463). It isalso contemplated that any of a variety of automated sequencingprocedures may be utilized when performing the subject assays (see, forexample Biotechniques (1995) 19:448), including sequencing by massspectrometry (see, for example PCT publication WO 94/16101; Cohen et al.(1996) Adv Chromatogr 36:127-162; and Griffin et al. (1993) Appl BiochemBiotechnol 38:147-159). It will be evident to one of skill in the artthat, for certain embodiments, the occurrence of only one, two or threeof the nucleic acid bases need be determined in the sequencing reaction.For instance, A-track or the like, e.g., where only one nucleic acid isdetected, can be carried out.

[0173] In a further embodiment, protection from cleavage agents (such asa nuclease, hydroxylamine or osmium tetroxide and with piperidine) canbe used to detect mismatched bases in RNA/RNA or RNA/DNA or DNA/DNAheteroduplexes (Myers, et al. (1985) Science 230:1242). In general, theart technique of “mismatch cleavage” starts by providing heteroduplexesformed by hybridizing (labeled) RNA or DNA containing the wild-typeallele with the sample. The double-stranded duplexes are treated with anagent which cleaves single-stranded regions of the duplex such as whichwill exist due to base pair mismatches between the control and samplestrands. For instance, RNA/DNA duplexes can be treated with RNase andDNA/DNA hybrids treated with S1 nuclease to enzymatically digest themismatched regions. In other embodiments, either DNA/DNA or RNA/DNAduplexes can be treated with hydroxylamine or osmium tetroxide and withpiperidine in order to digest mismatched regions. After digestion of themismatched regions, the resulting material is then separated by size ondenaturing polyacrylamide gels to determine the site of mutation. See,for example, Cotton et al (1988) Proc. Natl Acad Sci USA 85:4397; andSaleeba et al (1992) Methods Enzymol. 217:286-295. In a preferredembodiment, the control DNA or RNA can be labeled for detection.

[0174] In still another embodiment, the mismatch cleavage reactionemploys one or more proteins that recognize mismatched base pairs indouble-stranded DNA (so called “DNA mismatch repair” enzymes). Forexample, the mutY enzyme of E. coli cleaves A at G/A mismatches and thethymidine DNA glycosylase from HeLa cells cleaves T at G/T mismatches(Hsu et al. (1994) Carcinogenesis 15:1657-1662). According to anexemplary embodiment, a probe based on an allele of an IL-1 locushaplotype is hybridized to a CDNA or other DNA product from a testcell(s). The duplex is treated with a DNA mismatch repair enzyme, andthe cleavage products, if any, can be detected from electrophoresisprotocols or the like. See, for example, U.S. Pat. No. 5,459,039.

[0175] In other embodiments, alterations in electrophoretic mobilitywill be used to identify an IL-1 locus allele. For example, singlestrand conformation polymorphism (SSCP) may be used to detectdifferences in electrophoretic mobility between mutant and wild typenucleic acids (Orita et al. (1989) Proc Natl. Acad. Sci USA 86:2766, seealso Cotton (1993) Mutat Res 285:125-144; and Hayashi (1992) Genet AnalTech Appl 9:73-79). Single-stranded DNA fragments of sample and controlIL-1 locus alleles are denatured and allowed to renature. The secondarystructure of single-stranded nucleic acids varies according to sequence,the resulting alteration in electrophoretic mobility enables thedetection of even a single base change. The DNA fragments may be labeledor detected with labeled probes. The sensitivity of the assay may beenhanced by using RNA (rather than DNA), in which the secondarystructure is more sensitive to a change in sequence. In a preferredembodiment, the subject method utilizes heteroduplex analysis toseparate double stranded heteroduplex molecules on the basis of changesin electrophoretic mobility (Keen et al. (1991) Trends Genet 7:5).

[0176] In yet another embodiment, the movement of alleles inpolyacrylamide gels containing a gradient of denaturant is assayed usingdenaturing gradient gel electrophoresis (DGGE) (Myers et al. (1985)Nature 313:495). When DGGE is used as the method of analysis, DNA willbe modified to insure that it does not completely denature, for exampleby adding a GC clamp of approximately 40 bp of high-melting GC-rich DNAby PCR. In a further embodiment, a temperature gradient is used in placeof a denaturing agent gradient to identify differences in the mobilityof control and sample DNA (Rosenbaum and Reissner (1987) Biophys Chem265:12753).

[0177] Examples of other techniques for detecting alleles include, butare not limited to, selective oligonucleotide hybridization, selectiveamplification, or selective primer extension. For example,oligonucleotide primers may be prepared in which the known mutation ornucleotide difference (e.g., in allelic variants) is placed centrallyand then hybridized to target DNA under conditions which permithybridization only if a perfect match is found (Saiki et al. (1986)Nature 324:163); Saiki et al (1989) Proc. Natl Acad. Sci USA 86:6230).Such allele specific oligonucleotide hybridization techniques may beused to test one mutation or polymorphic region per reaction whenoligonucleotides are hybridized to PCR amplified target DNA or a numberof different mutations or polymorphic regions when the oligonucleotidesare attached to the hybridizing membrane and hybridized with labelledtarget DNA.

[0178] Alternatively, allele specific amplification technology whichdepends on selective PCR amplification may be used in conjunction withthe instant invention. Oligonucleotides used as primers for specificamplification may carry the mutation or polymorphic region of interestin the center of the molecule (so that amplification depends ondifferential hybridization) (Gibbs et al (1989) Nucleic Acids Res.17:2437-2448) or at the extreme 3′ end of one primer where, underappropriate conditions, mismatch can prevent, or reduce polymeraseextension (Prossner (1993) Tibtech 11:238. In addition it may bedesirable to introduce a novel restriction site in the region of themutation to create cleavage-based detection (Gasparini et al (1992) Mol.Cell Probes 6:1). It is anticipated that in certain embodimentsamplification may also be performed using Taq ligase for amplification(Barany (1991) Proc. Natl. Acad. Sci USA 88:189). In such cases,ligation will occur only if there is a perfect match at the 3′ end ofthe 5′ sequence making it possible to detect the presence of a knownmutation at a specific site by looking for the presence or absence ofamplification.

[0179] In another embodiment, identification of the allelic variant iscarried out using an oligonucleotide ligation assay (OLA), as described,e.g., in U.S. Pat. No. 4,998,617 and in Landegren, U. et al. ((1988)Science 241:1077-1080). The OLA protocol uses two oligonucleotides whichare designed to be capable of hybridizing to abutting sequences of asingle strand of a target. One of the oligonucleotides is linked to aseparation marker, e.g,. biotinylated, and the other is detectablylabeled. If the precise complementary sequence is found in a targetmolecule, the oligonucleotides will hybridize such that their terminiabut, and create a ligation substrate. Ligation then permits the labeledoligonucleotide to be recovered using avidin, or another biotin ligand.Nickerson, D. A. et al. have described a nucleic acid detection assaythat combines attributes of PCR and OLA (Nickerson, D. A. et al. (1990)Proc. Natl. Acad. Sci. USA 87:8923-27). In this method, PCR is used toachieve the exponential amplification of target DNA, which is thendetected using OLA.

[0180] Several techniques based on this OLA method have been developedand can be used to detect alleles of an IL-1 locus haplotype. Forexample, U.S. Pat. No. 5,593,826 discloses an OLA using anoligonucleotide having 3′-amino group and a 5′-phosphorylatedoligonucleotide to form a conjugate having a phosphoramidate linkage. Inanother variation of OLA described in Tobe et al. ((1996) Nucleic AcidsRes 24: 3728), OLA combined with PCR permits typing of two alleles in asingle microtiter well. By marking each of the allele-specific primerswith a unique hapten, i.e. digoxigenin and fluorescein, each OLAreaction can be detected by using hapten specific antibodies that arelabeled with different enzyme reporters, alkaline phosphatase orhorseradish peroxidase. This system permits the detection of the twoalleles using a high throughput format that leads to the production oftwo different colors.

[0181] Another embodiment of the invention is directed to kits fordetecting a predisposition for developing a cardiovascular disorder,either due to the occlusion of an artery or due to the formation offragile plaque, or due to the formation of restenosis. This kit maycontain one or more oligonucleotides, including 5′ and 3′oligonucleotides that hybridize 5′ and 3′ to at least one allele of anIL-1 locus haplotype. PCR amplification oligonucleotides shouldhybridize between 25 and 2500 base pairs apart, preferably between about100 and about 500 bases apart, in order to produce a PCR product ofconvenient size for subsequent analysis.

[0182] Particularly preferred primers for use in the diagnostic methodof the invention include SEQ ID Nos. 1-10.

[0183] The design of additional oligonucleotides for use in theamplification and detection of IL-1 polymorphic alleles by the method ofthe invention is facilitated by the availability of both updatedsequence information from human chromosome 2q13-which contains the humanIL-1 locus, and updated human polymorphism information available forthis locus. Suitable primers for the detection of a human polymorphismin these genes can be readily designed using this sequence informationand standard techniques known in the art for the design and optimizationof primers sequences. Optimal design of such primer sequences can beachieved, for example, by the use of commercially available primerselection programs such as Primer 2.1, Primer 3 or GeneFisher (See also,Nicklin M. H. J., Weith A. Duff G. W., “A Physical Map of the RegionEncompassing the Human Interleukin-1α, interleukin-1β, and Interleukin-1Receptor Antagonist Genes” Genomics 19: 382 (1995); Nothwang H. G., etal. “Molecular Cloning of the Interleukin-1 gene Cluster: Constructionof an Integrated YAC/PAC Contig and a partial transcriptional Map in theRegion of Chromosome 2q13” Genomics 41: 370 (1997); Clark, et al. (1986)Nucl. Acids. Res., 14:7897-7914 [published erratum appears in NucleicAcids Res., 15:868 (1987) and the Genome Database (GDB) project at theURL http://www.gdb.org).

[0184] For use in a kit, oligonucleotides may be any of a variety ofnatural and/or synthetic compositions such as syntheticoligonucleotides, restriction fragments, cDNAs, synthetic peptidenucleic acids (PNAs), and the like. The assay kit and method may alsoemploy labeled oligonucleotides to allow ease of identification in theassays. Examples of labels which may be employed include radio-labels,enzymes, fluorescent compounds, streptavidin, avidin, biotin, magneticmoieties, metal binding moieties, antigen or antibody moieties, and thelike.

[0185] The kit may, optionally, also include DNA sampling means. DNAsampling means are well known to one of skill in the art and caninclude, but not be limited to substrates, such as filter papers, theAmpliCard™ (University of Sheffield, Sheffield, England S10 2JF; Tarlow,J W, et al., J. of Invest. Dermatol. 103:387-389 (1994)) and the like;DNA purification reagents such as Nucleon™ kits, lysis buffers,proteinase solutions and the like; PCR reagents, such as 10×reactionbuffers, thermostable polymerase, dNTPs, and the like; and alleledetection means such as the HinfI restriction enzyme, allele specificoligonucleotides, degenerate oligonucleotide primers for nested PCR fromdried blood.

[0186] 4.3.3. Pharmacogenomics

[0187] Knowledge of the particular alleles associated with asusceptibility to developing a cardiovascular disorder, alone or inconjunction with information on other genetic defects contributing to acardiovascular disorder allows a customization of the prevention ortreatment in accordance with the individual's genetic profile, the goalof “pharmacogenomics”.

[0188] One approach to the prevention and treatment of a cardiovasculardisease relates to the identification of risk factors for the particulardisease.

[0189] For example, subjects having an allele 2 of any of the followingmarkers: IL-1A +4845 or IL-1B (+3954), or allele 1 of the followingmarkers: IL-1B (−511) or IL-1RN (+2018) or any nucleic acid sequence inlinkage disequilibrium with any of these alleles may have or bepredisposed to developing a cardiovascular disorder characterized by theformation of fragile plaque, may be predisposed to an increased risk ofmyocardial infarction, stroke, acute peripheral vascular blockage, andaneurysm formation in the mid-size to large arteries. These patients arealso predisposed to developing severe adult periodontitis.

[0190] Another approach to the treatment of cardiovascular diseasesrelates to interfering with the progression of the underlying disorder,ameliorating the symptoms and signs of the disease, or protecting atarget tissue so that the presence of a cardiovascular disorderaffecting the circulation of the tissue does not result in thedevelopment of clinical symptoms and signs related to that targettissue.

[0191] As an example, certain drugs have a stabilizing effect onatherosclerotic plaques or other beneficial effects on the sequelae offragile plaque disease. As examples, β-adrenergic receptor blockersreduce recurrence of myocardial infarction, angiotensin-convertingenzyme inhibitors reduce the incidence of myocardial infarction, certainantibiotics and antioxidants also have been shown to be effective instabilizing plaques. Drugs that have the capability of lowering lipids,such as 3-hydroxy-3methylglutaryl-coenzyme A reductase inhibitors(statins) are also important. Based on the disclosure of the pattern 1IL-1 genotype disclosed herein, these patients may respond better totherapeutics which are aimed at plaque stabilization rather thanrevascularization or other invasive techniques.

[0192] At the cellular level, lowering of serum cholesterol leads to adecrease in inflammatory cells within the artherosclerotic plaques. Atthe molecular level, lipid lowering has been shown to decreasemetalloproteinase activity in these plaques.

[0193] In one embodiment techniques such as gene therapy may be used tostabilize vulnerable plaque, for example, this could includeover-expression of tissue inhibitors of matrix metalloproteinases andanti-sense methods to block proinflammatory molecules.

[0194] On the other hand, subjects having an allele 1 of any of thefollowing markers: IL-1A +4845 or IL-1B (+3954), or allele 2 of thefollowing markers: IL-1B (−511) or IL-1RN (+2018) may respond better toparticular methods such as revascularization, or those methods thatalter the progression of intimal-medial arterial thickening.

[0195] Yet another approach to the management of cardiovasculardisorders and diseases comprises the management of conditions increasingrisk for cardio-vascular disorders.

[0196] Factors associated with the progression of atherosclerosisinclude Diabetes Mellitus, high blood pressure, Hypercholesterolemia,High lipoprotein-a, Obesity, and Smoking. Of these, the factors amenableto pharmacological intervention include: i) diabetes, ii) hypertension,and iii) dyslipidemias. Examples of lipid lowering drugs include: Anionexchange resins such as cholestyramine, colestipol; HMG CoA reductaseinhibitors or (statins) such as simvastatin, pracastatin, cerivastatin,fluvastatin, atorvastatin, lovastatin; Fibrates such as fenofibrate,bezafibrate, gemfibrozil, clofibrate, ciprofibrate; Nicotinic acid andanalogues: acipimox, nicofuranose; Probucol which increases non-receptormediated LDL clearance and decreases LDL oxidation; Fish oils such asmaxepa, Omacor; and Cholesterol absorption inhibitors such aspamaqueside, tiqueside.

[0197] Accordingly, therapeutics that address the particular molecularbasis of the disease in the subject may be developed based upon suchgenotype analysis. Thus, comparison of an individual's IL-1 profile tothe population profile for a cardiovascular disorder, permits theselection or design of drugs or other therapeutic regimens that areexpected to be safe and efficacious for a particular patient or patientpopulation (i.e., a group of patients having the same geneticalteration).

[0198] In addition, the ability to target populations expected to showthe highest clinical benefit, based on genetic profile can enable: 1)the repositioning of drugs already marketed for prevention or treatmentof cardiovascular disorder; 2) the rescue of drug candidates whoseclinical development has been discontinued as a result of safety orefficacy limitations, which are patient subgroup-specific; and 3) anaccelerated and less costly development for candidate therapeutics andmore optimal drug labeling (e.g. since measuring the effect of variousdoses of an agent on a vascular disorder causative mutation is usefulfor optimizing effective dose).

[0199] The treatment of an individual with a particular therapeutic canbe monitored by determining protein (e.g. IL-1α, IL-1β, or IL-1Ra), mRNAand/or transcriptional level. Depending on the level detected, thetherapeutic regimen can then be maintained or adjusted (increased ordecreased in dose). In a preferred embodiment, the effectiveness oftreating a subject with an agent comprises the steps of: (i) obtaining apreadministration sample from a subject prior to administration of theagent; (ii) detecting the level or amount of a protein, mRNA or genomicDNA in the preadministration sample; (iii) obtaining one or morepost-administration samples from the subject; (iv) detecting the levelof expression or activity of the protein, mRNA or genomic DNA in thepost-administration sample; (v) comparing the level of expression oractivity of the protein, mRNA or genomic DNA in the preadministrationsample with the corresponding protein, mRNA or genomic DNA in thepostadministration sample, respectively; and (vi) altering theadministration of the agent to the subject accordingly.

[0200] Cells of a subject may also be obtained before and afteradministration of a therapeutic to detect the level of expression ofgenes other than an IL-1 gene to verify that the therapeutic does notincrease or decrease the expression of genes which could be deleterious.This can be done, e.g., by using the method of transcriptionalprofiling. Thus, mRNA from cells exposed in vivo to a therapeutic andmRNA from the same type of cells that were not exposed to thetherapeutic could be reverse transcribed and hybridized to a chipcontaining DNA from numerous genes, to thereby compare the expression ofgenes in cells treated and not treated with the therapeutic.

[0201] 4.4 Therapeutics for Cardio Vascular Disorders and Diseases

[0202] Modulators of IL-1 (e.g. IL-1α, IL-1β or IL-1 receptorantagonist) or a protein encoded by a gene that is in linkagedisequilibrium with an IL-1 gene can comprise any type of compound,including a protein, peptide, peptidomimetic, small molecule, or nucleicacid. Preferred agonists include nucleic acids (e.g. encoding an IL-1protein or a gene that is up- or down-regulated by an IL-1 protein),proteins (e.g. IL-1 proteins or a protein that is up- or down-regulatedthereby) or a small molecule (e.g. that regulates expression or bindingof an IL-1 protein). Preferred antagonists, which can be identified, forexample, using the assays described herein, include nucleic acids (e.g.single (antisense) or double stranded (triplex) DNA or PNA andribozymes), protein (e.g. antibodies) and small molecules that act tosuppress or inhibit IL-1 transcription and/or protein activity.

[0203] 4.4.1. Effective Dose

[0204] Toxicity and therapeutic efficacy of such compounds can bedetermined by standard pharmaceutical procedures in cell cultures orexperimental animals, e.g., for determining The LD₅₀ (the dose lethal to50% of the population) and the Ed₅₀ (the dose therapeutically effectivein 50% of the population). The dose ratio between toxic and therapeuticeffects is the therapeutic index and it can be expressed as the ratioLD₅₀/ED₅₀. Compounds which exhibit large therapeutic indices arepreferred. While compounds that exhibit toxic side effects may be used,care should be taken to design a delivery system that targets suchcompounds to the site of affected tissues in order to minimize potentialdamage to uninfected cells and, thereby, reduce side effects.

[0205] The data obtained from the cell culture assays and animal studiescan be used in formulating a range of dosage for use in humans. Thedosage of such compounds lies preferably within a range of circulatingconcentrations that include the ED₅₀ with little or no toxicity. Thedosage may vary within this range depending upon the dosage formemployed and the route of administration utilized. For any compound usedin the method of the invention, the therapeutically effective dose canbe estimated initially from cell culture assays. A dose may beformulated in animal models to achieve a circulating plasmaconcentration range that includes the IC₅₀ (i.e., the concentration ofthe test compound which achieves a half-maximal inhibition of symptoms)as determined in cell culture. Such information can be used to moreaccurately determine useful doses in humans. Levels in plasma may bemeasured, for example, by high performance liquid chromatography.

[0206] 4.4.2. Formulation and Use

[0207] Compositions for use in accordance with the present invention maybe formulated in a conventional manner using one or more physiologicallyacceptable carriers or excipients. Thus, the compounds and theirphysiologically acceptable salts and solvates may be formulated, foradministration by, for example, injection, inhalation or insufflation(either through the mouth or the nose) or oral, buccal, parenteral orrectal administration.

[0208] For such therapy, the compounds of the invention can beformulated for a variety of loads of administration, including systemicand topical or localized administration. Techniques and formulationsgenerally may be found in Remmington's Pharmaceutical Sciences, MeadePublishing Co., Easton, Pa. For systemic administration, injection ispreferred, including intramuscular, intravenous, intraperitoneal, andsubcutaneous. For injection, the compounds of the invention can beformulated in liquid solutions, preferably in physiologically compatiblebuffers such as Hank's solution or Ringer's solution. In addition, thecompounds may be formulated in solid form and redissolved or suspendedimmediately prior to use. Lyophilized forms are also included.

[0209] For oral administration, the compositions may take the form of,for example, tablets or capsules prepared by conventional means withpharmaceutically acceptable excipients such as binding agents (e.g.,pregelatinised maize starch, polyvinylpyrrolidone or hydroxypropylmethylcellulose); fillers (e.g., lactose, microcrystalline cellulose orcalcium hydrogen phosphate); lubricants (e.g., magnesium stearate, talcor silica); disintegrants (e.g., potato starch or sodium starchglycolate); or wetting agents (e.g., sodium lauryl sulfate). The tabletsmay be coated by methods well known in the art. Liquid preparations fororal administration may take the form of, for example, solutions, syrupsor suspensions, or they may be presented as a dry product forconstitution with water or other suitable vehicle before use. Suchliquid preparations may be prepared by conventional means withpharmaceutically acceptable additives such as suspending agents (e.g.,sorbitol syrup, cellulose derivatives or hydrogenated edible fats);emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles(e.g., ationd oil, oily esters, ethyl alcohol or fractionated vegetableoils); and preservatives (e.g., methyl or propyl-p-hydroxybenzoates orsorbic acid). The preparations may also contain buffer salts, flavoring,coloring and sweetening agents as appropriate.

[0210] Preparations for oral administration may be suitably formulatedto give controlled release of the active compound. For buccaladministration the compositions may take the form of tablets or lozengesformulated in conventional manner. For administration by inhalation, thecompounds for use according to the present invention are convenientlydelivered in the form of an aerosol spray presentation from pressurizedpacks or a nebuliser, with the use of a suitable propellant, e.g.,dichlorodifluoromethane, trichlorofluoromethane,dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In thecase of a pressurized aerosol the dosage unit may be determined byproviding a valve to deliver a metered amount. Capsules and cartridgesof e.g., gelatin for use in an inhaler or insufflator may be formulatedcontaining a powder mix of the compound and a suitable powder base suchas lactose or starch.

[0211] The compounds may be formulated for parenteral administration byinjection, e.g., by bolus injection or continuous infusion. Formulationsfor injection may be presented in unit dosage form, e.g., in ampoules orin multi-dose containers, with an added preservative. The compositionsmay take such forms as suspensions, solutions or emulsions in oily oraqueous vehicles, and may contain formulating agents such as suspending,stabilizing and/or dispersing agents. Alternatively, the activeingredient may be in powder form for constitution with a suitablevehicle, e.g., sterile pyrogen-free water, before use.

[0212] The compounds may also be formulated in rectal compositions suchas suppositories or retention enemas, e.g., containing conventionalsuppository bases such as cocoa butter or other glycerides.

[0213] In addition to the formulations described previously, thecompounds may also be formulated as a depot preparation. Such longacting formulations may be administered by implantation (for examplesubcutaneously or intramuscularly) or by intramuscular injection. Thus,for example, the compounds may be formulated with suitable polymeric orhydrophobic materials (for example as an emulsion in an acceptable oil)or ion exchange resins, or as sparingly soluble derivatives, forexample, as a sparingly soluble salt. Other suitable delivery systemsinclude microspheres which offer the possibility of local noninvasivedelivery of drugs over an extended period of time. This technologyutilizes microspheres of precapillary size which can be injected via acoronary catheter into any selected part of the e.g. heart or otherorgans without causing inflammation or ischemia. The administeredtherapeutic is slowly released from these microspheres and taken up bysurrounding tissue cells (e.g. endothelial cells).

[0214] Systemic administration can also be by transmucosal ortransdermal means. For transmucosal or transdermal administration,penetrants appropriate to the barrier to be permeated are used in theformulation. Such penetrants are generally known in the art, andinclude, for example, for transmucosal administration bile salts andfusidic acid derivatives. In addition, detergents may be used tofacilitate permeation. Transmucosal administration may be through nasalsprays or using suppositories. For topical administration, the oligomersof the invention are formulated into ointments, salves, gels, or creamsas generally known in the art. A wash solution can be used locally totreat an injury or inflammation to accelerate healing.

[0215] The compositions may, if desired, be presented in a pack ordispenser device which may contain one or more unit dosage formscontaining the active ingredient. The pack may for example comprisemetal or plastic foil, such as a blister pack. The pack or dispenserdevice may be accompanied by instructions for administration.

[0216] 4.5 Assays to Identify Therapeutics for Cardio Vascular Disordersand Diseases

[0217] Based on the identification of mutations that cause or contributeto the development of a vascular disorder, the invention furtherfeatures cell-based or cell free assays, e.g., for identifying vasculardisorder therapeutics. In one embodiment, a cell expressing an IL-1receptor, or a receptor for a protein that is encoded by a gene which isin linkage disequilibrium with an IL-1 gene, on the outer surface of itscellular membrane is incubated in the presence of a test compound aloneor in the presence of a test compound and another protein and theinteraction between the test compound and the receptor or between theprotein (preferably a tagged protein) and the receptor is detected,e.g., by using a microphysiometer (McConnell et al. (1992) Science257:1906). An interaction between the receptor and either the testcompound or the protein is detected by the microphysiometer as a changein the acidification of the medium. This assay system thus provides ameans of identifying molecular antagonists which, for example, functionby interfering with protein-receptor interactions; as well as molecularagonist which, for example, function by activating a receptor.

[0218] Cellular or cell-free assays can also be used to identifycompounds which modulate expression of an IL-1 gene or a gene in linkagedisequilibrium therewith, modulate translation of an mRNA, or whichmodulate the stability of an mRNA or protein. Accordingly, in oneembodiment, a cell which is capable of producing an IL-1, or otherprotein is incubated with a test compound and the amount of proteinproduced in the cell medium is measured and compared to that producedfrom a cell which has not been contacted with the test compound. Thespecificity of the compound vis a vis the protein can be confirmed byvarious control analysis, e.g., measuring the expression of one or morecontrol genes. In particular, this assay can be used to determine theefficacy of antisense, ribozyme and triplex compounds.

[0219] Cell-free assays can also be used to identify compounds which arecapable of interacting with a protein, to thereby modify the activity ofthe protein. Such a compound can, e.g., modify the structure of aprotein thereby effecting its ability to bind to a receptor. In apreferred embodiment, cell-free assays for identifying such compoundsconsist essentially in a reaction mixture containing a protein and atest compound or a library of test compounds in the presence or absenceof a binding partner. A test compound can be, e.g., a derivative of abinding partner, e.g., a biologically inactive target peptide, or asmall molecule.

[0220] Accordingly, one exemplary screening assay of the presentinvention includes the steps of contacting a protein or functionalfragment thereof with a test compound or library of test compounds anddetecting the formation of complexes. For detection purposes, themolecule can be labeled with a specific marker and the test compound orlibrary of test compounds labeled with a different marker. Interactionof a test compound with a protein or fragment thereof can then bedetected by determining the level of the two labels after an incubationstep and a washing step. The presence of two labels after the washingstep is indicative of an interaction.

[0221] An interaction between molecules can also be identified by usingreal-time BIA (Biomolecular Interaction Analysis, Pharmacia BiosensorAB) which detects surface plasmon resonance (SPR), an opticalphenomenon. Detection depends on changes in the mass concentration ofmacromolecules at the biospecific interface, and does not require anylabeling of interactants. In one embodiment, a library of test compoundscan be immobilized on a sensor surface, e.g., which forms one wall of amicro-flow cell. A solution containing the protein or functionalfragment thereof is then flown continuously over the sensor surface. Achange in the resonance angle as shown on a signal recording, indicatesthat an interaction has occurred. This technique is further described,e.g., in BIAtechnology Handbook by Pharmacia.

[0222] Another exemplary screening assay of the present inventionincludes the steps of (a) forming a reaction mixture including: (i) anIL-1 or other protein, (ii) an appropriate receptor, and (iii) a testcompound; and (b) detecting interaction of the protein and receptor. Astatistically significant change (potentiation or inhibition) in theinteraction of the protein and receptor in the presence of the testcompound, relative to the interaction in the absence of the testcompound, indicates a potential antagonist (inhibitor). The compounds ofthis assay can be contacted simultaneously. Alternatively, a protein canfirst be contacted with a test compound for an appropriate amount oftime, following which the receptor is added to the reaction mixture. Theefficacy of the compound can be assessed by generating dose responsecurves from data obtained using various concentrations of the testcompound. Moreover, a control assay can also be performed to provide abaseline for comparison.

[0223] Complex formation between a protein and receptor may be detectedby a variety of techniques. Modulation of the formation of complexes canbe quantitated using, for example, detectably labeled proteins such asradiolabeled, fluorescently labeled, or enzymatically labeled proteinsor receptors, by immunoassay, or by chromatographic detection.

[0224] Typically, it will be desirable to immobilize either the proteinor the receptor to facilitate separation of complexes from uncomplexedforms of one or both of the proteins, as well as to accommodateautomation of the assay. Binding of protein and receptor can beaccomplished in any vessel suitable for containing the reactants.Examples include microtitre plates, test tubes, and micro-centrifugetubes. In one embodiment, a fusion protein can be provided which adds adomain that allows the protein to be bound to a matrix. For example,glutathione-S-transferase fusion proteins can be adsorbed ontoglutathione sepharose beads (Sigma Chemical, St. Louis, Mo.) orglutathione derivatized microtitre plates, which are then combined withthe receptor, e.g. an ³⁵S-labeled receptor, and the test compound, andthe mixture incubated under conditions conducive to complex formation,e.g. at physiological conditions for salt and pH, though slightly morestringent conditions may be desired. Following incubation, the beads arewashed to remove any unbound label, and the matrix immobilized andradiolabel determined directly (e.g. beads placed in scintillant), or inthe supernatant after the complexes are subsequently dissociated.Alternatively, the complexes can be dissociated from the matrix,separated by SDS-PAGE, and the level of protein or receptor found in thebead fraction quantitated from the gel using standard electrophoretictechniques such as described in the appended examples. Other techniquesfor immobilizing proteins on matrices are also available for use in thesubject assay. For instance, either protein or receptor can beimmobilized utilizing conjugation of biotin and streptavidin. Transgenicanimals can also be made to identify agonists and antagonists or toconfirm the safety and efficacy of a candidate therapeutic. Transgenicanimals of the invention can include non-human animals containing acardiovascular disorder causative mutation under the control of anappropriate endogenous promoter or under the control of a heterologouspromoter.

[0225] The transgenic animals can also be animals containing atransgene, such as reporter gene, under the control of an appropriatepromoter or fragment thereof. These animals are useful, e.g., foridentifying drugs that modulate production of an IL-1 protein, such asby modulating gene expression. Methods for obtaining transgenicnon-human animals are well known in the art. In preferred embodiments,the expression of the cardiovascular disorder causative mutation isrestricted to specific subsets of cells, tissues or developmental stagesutilizing, for example, cis-acting sequences that control expression inthe desired pattern. In the present invention, such mosaic expression ofa protein can be essential for many forms of lineage analysis and canadditionally provide a means to assess the effects of, for example,expression level which might grossly alter development in small patchesof tissue within an otherwise normal embryo. Toward this end,tissue-specific regulatory sequences and conditional regulatorysequences can be used to control expression of the mutation in certainspatial patterns. Moreover, temporal patterns of expression can beprovided by, for example, conditional recombination systems orprokaryotic transcriptional regulatory sequences. Genetic techniques,which allow for the expression of a mutation can be regulated viasite-specific genetic manipulation in vivo, are known to those skilledin the art.

[0226] The transgenic animals of the present invention all includewithin a plurality of their cells a cardiovascular disorder causativemutation transgene of the present invention, which transgene alters thephenotype of the “host cell”. In an illustrative embodiment, either thecre/loxP recombinase system of bacteriophage P1 (Lakso et al. (1992)PNAS 89:6232-6236; Orban et al. (1992) PNAS 89:6861-6865) or the FLPrecombinase system of Saccharomyces cerevisiae (O'Gorman et al. (1991)Science 251:1351-1355; PCT publication WO 92/15694) can be used togenerate in vivo site-specific genetic recombination systems. Crerecombinase catalyzes the site-specific recombination of an interveningtarget sequence located between loxP sequences. loxP sequences are 34base pair nucleotide repeat sequences to which the Cre recombinase bindsand are required for Cre recombinase mediated genetic recombination. Theorientation of loxP sequences determines whether the intervening targetsequence is excised or inverted when Cre recombinase is present(Abremski et al. (1984) J. Biol. Chem. 259:1509-1514); catalyzing theexcision of the target sequence when the loxP sequences are oriented asdirect repeats and catalyzes inversion of the target sequence when loxPsequences are oriented as inverted repeats.

[0227] Accordingly, genetic recombination of the target sequence isdependent on expression of the Cre recombinase. Expression of therecombinase can be regulated by promoter elements which are subject toregulatory control, e.g., tissue-specific, developmental stage-specific,inducible or repressible by externally added agents. This regulatedcontrol will result in genetic recombination of the target sequence onlyin cells where recombinase expression is mediated by the promoterelement. Thus, the activation of expression of the causative mutationtransgene can be regulated via control of recombinase expression.

[0228] Use of the cre/loxP recombinase system to regulate expression ofa causative mutation transgene requires the construction of a transgenicanimal containing transgenes encoding both the Cre recombinase and thesubject protein. Animals containing both the Cre recombinase and thecardio-vascular disorder causative mutation transgene can be providedthrough the construction of “double” transgenic animals. A convenientmethod for providing such animals is to mate two transgenic animals eachcontaining a transgene.

[0229] Similar conditional transgenes can be provided using prokaryoticpromoter sequences which require prokaryotic proteins to be simultaneousexpressed in order to facilitate expression of the transgene. Exemplarypromoters and the corresponding trans-activating prokaryotic proteinsare given in U.S. Pat. No. 4,833,080.

[0230] Moreover, expression of the conditional transgenes can be inducedby gene therapy-like methods wherein a gene encoding the transactivatingprotein, e.g. a recombinase or a prokaryotic protein, is delivered tothe tissue and caused to be expressed, such as in a cell-type specificmanner. By this method, the transgene could remain silent into adulthooduntil “turned on” by the introduction of the transactivator.

[0231] In an exemplary embodiment, the “transgenic non-human animals” ofthe invention are produced by introducing transgenes into the germlineof the non-human animal. Embryonal target cells at various developmentalstages can be used to introduce transgenes. Different methods are useddepending on the stage of development of the embryonal target cell. Thespecific line(s) of any animal used to practice this invention areselected for general good health, good embryo yields, good pronuclearvisibility in the embryo, and good reproductive fitness. In addition,the haplotype is a significant factor. For example, when transgenic miceare to be produced, strains such as C57BL/6 or FVB lines are often used(Jackson Laboratory, Bar Harbor, Me.). Preferred strains are those withH-2^(b), H-2^(d) or H-2^(q) haplotypes such as C57BL/6 or DBA/1. Theline(s) used to practice this invention may themselves be transgenics,and/or may be knockouts (i.e., obtained from animals which have one ormore genes partially or completely suppressed).

[0232] In one embodiment, the transgene construct is introduced into asingle stage embryo. The zygote is the best target for microinjection.In the mouse, the male pronucleus reaches the size of approximately 20micrometers in diameter which allows reproducible injection of 1-2 pl ofDNA solution. The use of zygotes as a target for gene transfer has amajor advantage in that in most cases the injected DNA will beincorporated into the host gene before the first cleavage (Brinster etal. (1985) PNAS 82:4438-4442). As a consequence, all cells of thetransgenic animal will carry the incorporated transgene. This will ingeneral also be reflected in the efficient transmission of the transgeneto offspring of the founder since 50% of the germ cells will harbor thetransgene.

[0233] Normally, fertilized embryos are incubated in suitable mediauntil the pronuclei appear. At about this time, the nucleotide sequencecomprising the transgene is introduced into the female or malepronucleus as described below. In some species such as mice, the malepronucleus is preferred. It is most preferred that the exogenous geneticmaterial be added to the male DNA complement of the zygote prior to itsbeing processed by the ovum nucleus or the zygote female pronucleus. Itis thought that the ovum nucleus or female pronucleus release moleculeswhich affect the male DNA complement, perhaps by replacing theprotamines of the male DNA with histones, thereby facilitating thecombination of the female and male DNA complements to form the diploidzygote. Thus, it is preferred that the exogenous genetic material beadded to the male complement of DNA or any other complement of DNA priorto its being affected by the female pronucleus. For example, theexogenous genetic material is added to the early male pronucleus, assoon as possible after the formation of the male pronucleus, which iswhen the male and female pronuclei are well separated and both arelocated close to the cell membrane. Alternatively, the exogenous geneticmaterial could be added to the nucleus of the sperm after it has beeninduced to undergo decondensation. Sperm containing the exogenousgenetic material can then be added to the ovum or the decondensed spermcould be added to the ovum with the transgene constructs being added assoon as possible thereafter.

[0234] Introduction of the transgene nucleotide sequence into the embryomay be accomplished by any means known in the art such as, for example,microinjection, electroporation, or lipofection. Following introductionof the transgene nucleotide sequence into the embryo, the embryo may beincubated in vitro for varying amounts of time, or reimplanted into thesurrogate host, or both. In vitro incubation to maturity is within thescope of this invention. One common method in to incubate the embryos invitro for about 1-7 days, depending on the species, and then reimplantthem into the surrogate host.

[0235] For the purposes of this invention a zygote is essentially theformation of a diploid cell which is capable of developing into acomplete organism. Generally, the zygote will be comprised of an eggcontaining a nucleus formed, either naturally or artificially, by thefusion of two haploid nuclei from a gamete or gametes. Thus, the gametenuclei must be ones which are naturally compatible, i.e., ones whichresult in a viable zygote capable of undergoing differentiation anddeveloping into a functioning organism. Generally, a euploid zygote ispreferred. If an aneuploid zygote is obtained, then the number ofchromosomes should not vary by more than one with respect to the euploidnumber of the organism from which either gamete originated.

[0236] In addition to similar biological considerations, physical onesalso govern the amount (e.g., volume) of exogenous genetic materialwhich can be added to the nucleus of the zygote or to the geneticmaterial which forms a part of the zygote nucleus. If no geneticmaterial is removed, then the amount of exogenous genetic material whichcan be added is limited by the amount which will be absorbed withoutbeing physically disruptive. Generally, the volume of exogenous geneticmaterial inserted will not exceed about 10 picoliters. The physicaleffects of addition must not be so great as to physically destroy theviability of the zygote. The biological limit of the number and varietyof DNA sequences will vary depending upon the particular zygote andfunctions of the exogenous genetic material and will be readily apparentto one skilled in the art, because the genetic material, including theexogenous genetic material, of the resulting zygote must be biologicallycapable of initiating and maintaining the differentiation anddevelopment of the zygote into a functional organism.

[0237] The number of copies of the transgene constructs which are addedto the zygote is dependent upon the total amount of exogenous geneticmaterial added and will be the amount which enables the genetictransformation to occur. Theoretically only one copy is required;however, generally, numerous copies are utilized, for example,1,000-20,000 copies of the transgene construct, in order to insure thatone copy is functional. As regards the present invention, there willoften be an advantage to having more than one functioning copy of eachof the inserted exogenous DNA sequences to enhance the phenotypicexpression of the exogenous DNA sequences.

[0238] Any technique which allows for the addition of the exogenousgenetic material into nucleic genetic material can be utilized so longas it is not destructive to the cell, nuclear membrane or other existingcellular or genetic structures. The exogenous genetic material ispreferentially inserted into the nucleic genetic material bymicroinjection. Microinjection of cells and cellular structures is knownand is used in the art.

[0239] Reimplantation is accomplished using standard methods. Usually,the surrogate host is anesthetized, and the embryos are inserted intothe oviduct. The number of embryos implanted into a particular host willvary by species, but will usually be comparable to the number of offspring the species naturally produces.

[0240] Transgenic offspring of the surrogate host may be screened forthe presence and/or expression of the transgene by any suitable method.Screening is often accomplished by Southern blot or Northern blotanalysis, using a probe that is complementary to at least a portion ofthe transgene. Western blot analysis using an antibody against theprotein encoded by the transgene may be employed as an alternative oradditional method for screening for the presence of the transgeneproduct. Typically, DNA is prepared from tail tissue and analyzed bySouthern analysis or PCR for the transgene. Alternatively, the tissuesor cells believed to express the transgene at the highest levels aretested for the presence and expression of the transgene using Southernanalysis or PCR, although any tissues or cell types may be used for thisanalysis.

[0241] Alternative or additional methods for evaluating the presence ofthe transgene include, without limitation, suitable biochemical assayssuch as enzyme and/or immunological assays, histological stains forparticular marker or enzyme activities, flow cytometric analysis, andthe like. Analysis of the blood may also be useful to detect thepresence of the transgene product in the blood, as well as to evaluatethe effect of the transgene on the levels of various types of bloodcells and other blood constituents.

[0242] Progeny of the transgenic animals may be obtained by mating thetransgenic animal with a suitable partner, or by in vitro fertilizationof eggs and/or sperm obtained from the transgenic animal. Where matingwith a partner is to be performed, the partner may or may not betransgenic and/or a knockout; where it is transgenic, it may contain thesame or a different transgene, or both. Alternatively, the partner maybe a parental line. Where in vitro fertilization is used, the fertilizedembryo may be implanted into a surrogate host or incubated in vitro, orboth. Using either method, the progeny may be evaluated for the presenceof the transgene using methods described above, or other appropriatemethods.

[0243] The transgenic animals produced in accordance with the presentinvention will include exogenous genetic material. Further, in suchembodiments the sequence will be attached to a transcriptional controlelement, e.g., a promoter, which preferably allows the expression of thetransgene product in a specific type of cell.

[0244] Retroviral infection can also be used to introduce the transgeneinto a non-human animal. The developing non-human embryo can be culturedin vitro to the blastocyst stage. During this time, the blastomeres canbe targets for retroviral infection (Jaenich, R. (1976) PNAS73:1260-1264). Efficient infection of the blastomeres is obtained byenzymatic treatment to remove the zona pellucida (Manipulating the MouseEmbryo, Hogan eds. (Cold Spring Harbor Laboratory Press, Cold SpringHarbor, 1986). The viral vector system used to introduce the transgeneis typically a replication-defective retrovirus carrying the transgene(Jahner et al. (1985) PNAS 82:6927-6931; Van der Putten et al. (1985)PNAS 82:6148-6152). Transfection is easily and efficiently obtained byculturing the blastomeres on a monolayer of virus-producing cells (Vander Putten, supra; Stewart et al. (1987) EMBO J. 6:383-388).Alternatively, infection can be performed at a later stage. Virus orvirus-producing cells can be injected into the blastocoele (Jahner etal. (1982) Nature 298:623-628). Most of the founders will be mosaic forthe transgene since incorporation occurs only in a subset of the cellswhich formed the transgenic non-human animal. Further, the founder maycontain various retroviral insertions of the transgene at differentpositions in the genome which generally will segregate in the offspring.In addition, it is also possible to introduce transgenes into the germline by intrauterine retroviral infection of the midgestation embryo(Jahner et al. (1982) supra).

[0245] A third type of target cell for transgene introduction is theembryonal stem cell (ES). ES cells are obtained from pre-implantationembryos cultured in vitro and fused with embryos (Evans et al. (1981)Nature 292:154-156; Bradley et al. (1984) Nature 309:255-258; Gossler etal. (1986) PNAS 83: 9065-9069; and Robertson et al. (1986) Nature322:445-448). Transgenes can be efficiently introduced into the ES cellsby DNA transfection or by retrovirus-mediated transduction. Suchtransformed ES cells can thereafter be combined with blastocysts from anon-human animal. The ES cells thereafter colonize the embryo andcontribute to the germ line of the resulting chimeric animal. For reviewsee Jaenisch, R. (1988) Science 240:1468-1474.

[0246] The present invention is further illustrated by the followingexamples which should not be construed as limiting in any way. Thecontents of all cited references (including literature references,issued patents, published patent applications as cited throughout thisapplication) are hereby expressly incorporated by reference. Thepractice of the present invention will employ, unless otherwiseindicated, conventional techniques that are within the skill of the art.Such techniques are explained fully in the literature. See, for example,Molecular Cloning A Laboratory Manual, (2nd ed., Sambrook, Fritsch andManiatis, eds., Cold Spring Harbor Laboratory Press: 1989); DNA Cloning,Volumes I and II (D. N. Glover ed., 1985); Oligonucleotide Synthesis (M.J. Gait ed., 1984); U.S. Pat. Nos. 4,683,195; 4,683,202; and NucleicAcid Hybridization (B. D. Hames & S. J. Higgins eds., 1984).

5. EXAMPLES Example 1

[0247] Markers for Single Vessel Coronary Artery Disease

[0248] The objective of this study was to determine if patients with anearly form of coronary artery atherosclerosis, i.e., single vesselcoronary artery disease, were more likely to have specific alleles inthe following genes: IL-1A (−889 marker), IL-1B (−511 and +3954markers), IL-1RN (VNTR marker) or TNFα (−308 marker). Multiple vesseldisease generally represents a later stage of the disease that mayinvolve many factors which could complicate data interpretation.Therefore, patients who presented with a complaint of chest pain wereevaluated by a cardiologist, and those with angiographic evidence ofsignificant atherosclerosis in more than one coronary artery wereexcluded from analysis.

[0249] Patient Cohorts: Angiography from either the femoral or brachialartery was performed using conventional techniques. Of the patientsexamined, eighty-five (85) had no obvious luminal irregularities byangiography and were classified as controls having angiographicallynormal coronary arteries. A patient was classified as having singlevessel disease if one of three epicardial coronary vessels containing anepicardial stenosis causing 50% reduction in luminal diameter, asassessed by eye. Fifty-eight (58) patients were found to have singlevessel coronary artery disease. Patients with multiple vessel diseasewere excluded. Both control and single vessel disease groups hadcomparable mean ages, 57.6±10.4 years and 56.4±9.4 years; respectively.The male to female ratio in the control group was 1:1.7 and 2.6:1 in thediseased group.

[0250] General Methods: Reactions and manipulations involving nucleicacid techniques, unless stated otherwise, were performed as generallydescribed in Sambrook, et al., Molecular Cloning: A Laboratory Manual,Cold Spring Harbor Laboratory Press (1989). Polymerase chain reaction(PCR) was carried out generally as described in PCR Protocols: A Guideto Methods and Applications, Academic Press, San Diego, Calif. (1990).Genotyping methodology was as generally described in U.S. Pat. Nos.4,666,828; 4,683,202; 4,801,531; 5,192,659; and 5,272,057 and McDowell,et al., Arthritis & Rheumatism, 38(2):221-8 (1995).

[0251] DNA Preparation: DNA was extracted from whole blood using amodification of the salt-out method (Nucleon II™, Scotlab, UK).

[0252] Genotyping IL-1RN: Alleles associated with the IL-1RN gene werepreviously described by Tarlow, et al., Human Genetics, 91:403-4 (1993).Enzymes used in PCR were from Promega (UK) and thermocyclers were eitherMJ Research DNA Engine or Biometra. The following primers were producedin an ABI DNA synthesizer: 5′CTCAGCAACACTCCTAT 3′ (SEQ ID No. 1)5′TCCTGGTCTGCAGGTAA 3′ (SEQ ID No. 2)

[0253] PCR amplification was performed with a final magnesiumconcentration of 1.75 mM and a cycling protocol of 1 cycle at 96° C. for1 minute; 30 cycles of [94° C. for 1 minute, 60° C. for 1 minute, and70° C. for 1 minute]; and 1 cycle at 70° C. for 2 minutes. Following PCRthe different alleles were electrophoresed on 2% agarose gel stainedwith ethidium bromide and visualized and identified under uv light.Negative controls without DNA were performed in each experiment.

[0254] Intron 2 of the IL-1RN gene contains a variable number tandemrepeat (VNTR) region that gives rise to five (5) alleles as follows:

[0255] Allele 1 contains four repeats and displays a 412 bp PCR product;

[0256] Allele 2 contains two repeats and displays a 240 bp PCR product;

[0257] Allele 3 contains three repeats and displays a 326 bp PCRproduct;

[0258] Allele 4 contains five repeats and displays a 498 bp PCR product;and

[0259] Allele 5 contains six repeats and displays a 584 bp PCR product.

[0260] Genotyping IL-1B (−511)

[0261] The −511 marker of IL-1B was described by diGiovine, Hum. Molec.Genet., 1(6):450 (1992). The single base variation (C/T) marker at IL-1Bbase −511 was identified on the basis of an AvaI site on allele 1(C),and a Bsu36I site on allele 2(T). PCR was performed with 1 cycle at 95°C. for 2 minutes, 35 cycles at [95° C. for 1 minute, 53° C. for 1minute, and 74° C. for 1 minute] and 1 cycle at 74° C. for 4 minutes.Analysis of the PCR products was by restriction enzyme digestion withAvaI and Bsu36I at 37° C. for 8 hours followed by size analysis with 8%PAGE. The following primers were produced in an ABI DNA synthesizer(Clark, et al., Nucl. Acids. Res., 14:7897-7914 (1986) [publishederratum appears in Nucleic Acids Res., 15(2):868 (1987)]; GENBANKX04500): 5′TGGCATTGATCTGGTTCATC 3′ (SEQ ID No: 5) (−702/−682)5′GTTTAGGAATCTTCCCACTT 3′ (SEQ ID No: 6) (−417/−397)

[0262] Results: There was no significant difference between the controland diseased patients in the frequency of different alleles in the genesfor IL-1A (−889 marker), IL-1B (+3954 marker) or TNFα (−308 marker).However, allele 2 of the VNTR marker in the IL-1RN gene wassignificantly over-represented in the single vessel disease patients,41% versus 22% in controls. It is estimated that individuals with atleast one copy of allele 2 are 2.44 times as likely to have singlevessel coronary artery disease than those who are negative for allele 2(odds Ratio=2.44, p=0.003, 95% confidence interval=1.35-4.43).

[0263] In addition, individuals who had two copies, i.e., werehomozygous for allele 2 in IL-1RN, were 5.36 times as likely to havesingle vessel coronary artery disease than those who were negative forallele 2 (odds Ratio=5.36, p=0.005, 95% confidence interval=1.6-17.97).

[0264] Carriage of one copy of allele 2 of the −511 marker of the IL-1Bgene was increased in single vessel coronary disease to 52% comparedwith 38% in controls. It is estimated that individuals with at least onecopy of allele 2 are 1.74 times as likely to have single vessel diseasethan those who are negative for allele 2 (Odds Ratio=1.74, p=0.1, 95%confidence interval=0.86-3.52).

[0265] These findings indicate that allele 2 of the IL-1RN gene is amarker for susceptibility to the development of coronary arteryocclusive disease, manifested as single-vessel stenosis. This allele isassociated with an increased risk of coronary artery disease of 2.4 to5.4 times, depending on whether there in one copy (heterozygous) or twocopies (homozygous) of the disease-associated allele. The influence ofthis allele on risk for coronary artery disease is shown in Table 1relative to other common risk factors.

[0266] Additionally, an allele for the IL-1B gene was discovered to beassociated with single vessel coronary artery disease. This allele isassociated with an increased risk of coronary artery disease of 1.74times. TABLE 1 Increased Risk for Coronary Risk Factor Artery DiseaseSmoking (1 pack/day) 2.5 Sedentary lifestyle 1.9 Severe obesity (women)3.3 Hypertension 2.1 High cholesterol (>240) 2.4 IL-1RN (VNTR) allele2-heterozygous 2.4 IL-1RN (VNTR) allele 2-homozygous 5.4 IL-1B (−511)allele 2 1.74-1.92

Example 2

[0267] Markers for Multiple Vessel Coronary Artery Disease

[0268] The objective of this study was to determine if patients with alater or more diffuse form of coronary artery atherosclerosis, i.e.,multiple vessel coronary artery disease, were more likely to havespecific alleles in the genes of the IL-1 gene cluster or TNFα.

[0269] Patient Coborts: Patient cohorts were determined as in Example 1,except that a patient was classified as having multiple vessel diseaseif more than one epicardial coronary vessel contained an epicardialstenosis causing >50% reduction in luminal diameter, as assessed by eye.Of the patients examined, 86 were classified as controls havingangiographically normal coronary arteries and 315 patients were found tohave multiple vessel coronary artery disease. Both controls and singlevessel disease groups had comparable mean ages, 57.6±10.4 years and60.8±1.13 years respectively. The male to female ratio in the controlgroup was 1:1:7 and 3.7:1 in the diseased group.

[0270] General Methods: Reactions and methods were as in Example 1.

[0271] Results: There was no significant difference between the controland diseased patients in the frequency of different alleles in the genesfor IL-1A (−889 marker), IL-1B (+3954 marker), and IL-1RN (VNTR marker).However, carriage of one copy of the Bsu36I allele (allele 2) of the−511 marker of the IL-1B gene was increased in the multiple vesseldisease patients, 54% versus 38% in controls. It is estimated thatindividuals with at least one copy of allele 2 of the −511 marker are1.92 times as likely to have multiple vessel coronary artery diseasethan those who are negative for allele 2 (Odds Ratio+1.92, p=0.009, 95%confidence interval=1.17-3.16). There appears to be no dose effect, inthis population at least, for the −511 marker.

[0272] In summary, an allele for the IL-1B gene was discovered to beassociated with multiple vessel coronary artery disease. This allele isassociated with an increased risk of coronary artery disease of 1.92times.

[0273] Single vessel and multiple vessel coronary artery disease eachappear to be linked with different genes of the IL-1 gene cluster. Thismay arise as a true biological distinction, where IL-1 RA modulatesIL-1β effects in such a way as to produce the single vessel phenotype.Alternatively, it may be that both genes are, in fact, associated withcoronary artery disease as a whole and that the associations observedhere result from the way this particular population exhibited coronaryartery disease. With either interpretation, a strong association betweenIL-1 biology and coronary artery disease has been established.

Example 3

[0274] Association of Interleukin-1 Gene Variants and Carotid ArterialWall Thickness

[0275] The association between carotid intimal medial wall thickness(IMT) and four basic biallelic markers (IL-1A (+4845), IL-1B (+3954),IL-1RN (+2018)) in the interleukin-1 (IL-1) gene cluster on chromosome 2was investigated among participants in the Atherosclerosis Risk inCommunities (ARIC) Study, a cohort of 15,792 men and women 45-64 yearsof age selected from four U.S. communities. Far wall thickness wasmeasured by B-mode ultrasound and analyzed using a cutpoint for elevatedaverage IMT (≧1 mm) chosen a priori to identify individuals at greatestrisk of cardiovascular disease. After excluding those with a history ofcardiovascular disease, a stratified random sample of 252 AfricanAmericans and 924 Caucasians was genotyped. Among African Americans,carriers of the less common allele (allele 2) of IL-1RN (+2018) weremore likely than non-carriers to have average IMT ≧1 mm (16% vs 5%p=0.04) in a basic model adjusting for age, gender and study center.Among Caucasians, the adjusted proportion of individuals with elevatedIMT was also higher in those carrying IL-1RN (+2018) allele 2 (9% vs.6%), but this difference was not statistically significant (p=0.10).There were no associations between the IL-1A (+4845), IL-1B (+3954) orIL-1B (−511) variants and carotid IMT in either ethnic group.

Example 4

[0276] IL-1 Genotypes Associated with Plaque Formation and IncreasedPlaque Fragility

[0277] Polymorphisms in the gene for IL-1 receptor antagonist and thelinked IL-1B(−511) gene are strongly associated with the presence oflarge (>50% occlusion of the vessel) plaques in the coronary arteriesand early atherosclerotic changes in the carotid artery wall (ARICdata). These data suggest that the genetic polymorphism pattern thatinvolves IL-1RN(+2018) allele 2 and/or IL-1B(−511) allele 2 ispredictive of large, occluding plaques.

[0278] Certain IL-1 genotypes are associated with increased risk forclinical events such as thrombosis and embolism. We propose that allele2 in either or both of the loci IL-1A(+4845) and IL-1B(+3954) would beexpected to increase the inflammatory response and therefore increaseplaque fragility and risk for clinical events such as thrombosis andembolism. This risk may be greatest in individuals with low levels ofcholesterol, since higher levels would be expected to activate themaximal inflammatory response even in IL-1 wild-types (e.g.IL-1A(+4845)=1.1 and IL-1B(+3954)=1.1).

[0279] We propose that the genotypes associated with larger occlusiveplaques, i.e. IL-1RN(+2018) allele 2 or IL-1B(−511) allele 2, would bepredictive of lower risk for plaque fragility.

[0280] Out of approximately 15,000 healthy individuals followedlongitudinally for clinical events (ARIC), 370 thrombotic or embolicevents were documented. A group of approximately 900 randomizedstratified controls were selected for comparison.

[0281] Allele 2 at IL-1A(+4845) and IL-1B(+3954) Influence FragilePlaque-Related Clinical Events:

[0282] For cases with LDL<130 (n==535)

[0283] IL-1A(+4845) genotype 2.2: Odds ratio(OR+95%CI) for clinicalevent=3.03(0.96-9.1); p=0.059

[0284] For cases with Total Cholesterol <200 (n=425) IL-1A(+4845)genotype 2.2: OR = 6.25 (1.69-20.00); p = 0.006 IL-1B(+3954) genotype1.2 or 2.2: OR = 2.58 (1.25-5.31); p = 0.010

[0285] Allele 2 at IL-1RN(+2018) is Inversely Related to FragilePlaque-Related Clinical Events, thereby Suggesting a Stabilization ofAtherosclerotic Plaque:

[0286] For all cases (n=1214)

[0287] IL-1RN(+2018) genotype 1.2 or 2.2: OR=0.65(0.43-0.96); p=0.031

[0288] For LDL >160(n=343)

[0289] IL-1RN(+2018) genotype 1.2 or 2.2: OR=0.33(0.14-0.73); p=0.058

[0290] For Total Cholesterol >240 (n=307)

[0291] IL-1RN(+2018) genotype 1.2 or 2.2: OR=0.28(0.11-0.68); p=0.054

Example 5

[0292] The IL-1 Composite Genotype that is Consistent with HaplotypePattern 1 is Associated with Periodontitis and the IL-1 Genotype that isConsistent with Haplotype Pattern 2 is Associated with OcclusiveCardiovascular Disease

[0293] The association between periodontitis, cardiovascular disease andfour basic biallelic markers (IL-1A (+4845), IL-1B (+3954), IL-1B(−511), and IL-1RN (+2018)) in the interleukin-1 (IL-1) gene cluster onchromosome 2 was investigated.

[0294] Two haplotype patterns may be defined by four polymorphic loci inthe IL-1 gene cluster as shown in Table 2 (IL-1A(+4845), IL-1B(+3954),IL-1B (−511), IL-1RN(+2018)). One pattern includes allele 2 at both theIL-1A (+4845) and at the IL-1B (+3954) loci. The other pattern includesallele 2 at both the IL-1B(−511), and at the IL-1RN(+2018) loci. TABLE 2IL-1A IL-1B Haplotypes (+4845) (+3954) IL-1B (−511) IL-1RN (+2018)Pattern 1 Allele 2 Allele 2 Allele 1 Allele 1 Pattern 2 Allele 1 Allele1 Allele 2 Allele 2

[0295] The haplotype pattern indicates that when allele 2 is found atone locus, it is highly likely that it will be found at other loci.Previous data (Cox et al. (1998) Am. J. Hum. Genet. 62:1180-1188)indicate that when allele 2 is found at the IL-1A (+4845) locus allele 2will also be present at the IL-1B (+3954) locus approximately 80% of thetime. Haplotype patterns are relevant only for a single copy of achromosome. Since there are two copies of chromosome 2 and standardgenotyping procedures are unable to identify on which chromosome copy aspecific allele is found, special statistical programs are used to inferhaplotype patterns from the genotype pattern that is determined.

[0296] The distribution of these genetic patterns was evaluated in a newpopulation that was part of a study of atherosclerosis (Pankow et al.(1999) The ARIC study. European Atherosclerosis Society Annual Meeting,Abstract, #646). In this population (N=1,368), IL-1A(+4845) genotype 2.2was found in 10.2% of the subjects. However, in the subjects withgenotype IL-1B(+3954)=2.2(N-95), the IL-1A (+4845) genotype 2.2 wasfound in 71.6% of the subjects. This indicates that allele 2 at IL-1A(+4845) is inherited together with allele 2 at IL-1B (3954) at a muchhigher rate than one would expect given the distribution of each ofthese markers in the population. Similar data exists for allele 2 at the2 loci that are characteristic of Pattern 2. In addition, when genotypePattern 1 is found it is highly unlikely that allele 2 will be presentat either of the loci that are characteristic of the other pattern.

[0297] The two genotype patterns are also associated with specificdifferences in the functional biology of interleukin-1. For example,peripheral monocytes from individuals with one or two copies of allele 2at IL-1B (+3954) produced 2 to 4 times as much IL-1β when stimulatedwith LPS as monocytes from individuals who have the genotype patternIL-1B (+3954)=1.1 (DiGiovini, F S et al. (1995) Cytokine, 7:606).Similar data have recently been reported for peripheral bloodpolymorphonuclear leukocytes isolated from individuals with severeperiodontitis (Gore, E A et al. (1998) J. Clin. Periodontol., 25:781).In addition gingival crevice fluid (GCF) from subjects with thecomposite genotypes indicative of Pattern 1 have 2 to 3 times higherlevels of IL-1β than GCF from individuals who are negative for thosegenotypes (Engelbretson, S P et al. (1999) J. Periodontol., in press).There are also data indicating that for Pattern 2, allele 2 at IL-1RN+2018 is associated with decreased levels of IL-1 receptor antagonistprotein. Thus, Pattern 1 genotypes appear to be associated withincreased IL-1 agonists, and Pattern 2 appears to be associated withdecreased levels of IL-1 receptor antagonist.

[0298] The composite IL-1 genotypes that are consistent with Pattern 1are associated with increased susceptibility to severe adultperiodontitis (Kornman, K S et al. (1997), supra; Gore, E A et al.(1998), supra; McGuire, M K et al. (1999) J. Periodontol., in press;McDevitt, M J et al. (1999) J. Periodontol., in press). One aspect ofthe IL-1 genotype influence on periodontitis appears to be anenhancement of the subgingival levels of specific bacterial complexesthat include accepted periodontal pathogens (Socransky, S S et al.(1999) IADR Annual Meeting, Abstract#3600). Pattern 1 genotypes werenot, however, associated with increased risk for occlusivecardiovascular disease. In data from the Atherosclerosis Risk inCommunities (ARIC) study that was presented by Pankow and co-workers(see Pankow et al., supra), individuals with ultrasound measurements ofcarotid wall intima-medial thickness (IMT) that were indicative ofocclusive cardiovascular disorders were compared to a stratified randomcontrol population for IL-1 gene polymorphisms. Neither IL-1A (+4845) orIL-1B (+3954) showed any association with risk for high IMT.

[0299] Genotypes that are characteristic of pattern 2 have recently beenassociated with increased susceptibility to occlusive coronary arterydisease, but not increased risk for periodontitis. In a report oncoronary artery disease, patients with angiographic evidence of coronarystenoses were significantly more likely to be carriers of allele 2 ateither the IL-1RN (+2018) locus or the IL-1B (−511) locus (see Franciset al., supra). Both loci are characteristic of the haplotype Pattern 2.In the ARIC study, as discussed above, carriage of IL-1RN (+2018) allele2 in African-Americans with high IMT measurements was significantlyhigher than ethnically matched controls. In Caucasians with high IMTmeasurements the carriage of one copy of allele 2 at IL-1RN (+2018) wassignificantly greater than in controls, however individuals homozygousat this locus were not different from controls. It should be noted thatthe prevalence of individuals homozygous for allele 2 at IL-1RN (+2018)in Caucasians in the study was substantially lower than that observed inother populations.

[0300] When individuals with periodontitis and gingival health wereevaluated for genotype patterns consistent with Pattern 1 and Pattern 2,individuals with severe adult periodontitis were found to have apredominance of genotypes consistent with Pattern 1, whereas individualswith a healthy periodontal condition had genotype patterns that weredominated by neither Pattern 1 nor Pattern 2. It appears therefore thatIL-1 genotypes consistent with the haplotype Pattern 1 are associatedwith severe periodontitis and plaque fragility disorders and notocclusive cardiovascluar diseases whereas IL-1 genotypes consistent withthe haplotype Pattern 2 are associated with occlusive cardiovasculardiseases but not periodontitis or plaque fragility. One mechanism may bethat IL-1 genotype Pattern 1 directly influences plaque fragility;another mechanism may be that Pattern 1 influences periodontitisdirectly, which may lead to indirect influences on cardiovasculardisease through the periodontal micororganisms found as part of the oralchronic inflammatory process. Another mechanism may be that IL-1genotype Pattern 2 directly influences cardiovascular occlusivedisorders but has no influence on periodontitis. It is thus likely thatIL-1 genetic polymorphisms can influence both cardiovascular disease andsevere periodontitis, by a common underlying mechanism that directlyalters the immunoinflammatory responses in both diseases in an identicalfashion and by an indirect mechanism that enhances the oral bacterialload and then influences cardiovascular disease. The IL-1 genotypes thatare consistent with haplotype Pattern 1 may influence the associationbetween periodontidis and cardiovascular disease in one segment of thepopulation by amplifying both the immuno-inflammatory response and thesubgingival bacterial load.

Example 5

[0301] The Mayo Clinic Study

[0302] Study design. Patients 18 to 75 years of age undergoingclinically-indicated coronary angiography at Mayo Clinic, Rochester,Minn. were considered for this study. Patients were ineligible forinclusion if they had diabetes mellitus requiring therapy, a smokinghistory >50 pack years, prior or planned organ transplantation,pregnancy, prior percutaneous or surgical coronary revascularization,active bleeding or hemoglobin less than 8 g/dL, receipt of a bloodtransfusion within 30 days, hemodynamic instability, infection withhuman immunodeficiency virus, renal failure requiring dialysis, and ahistory of radiation therapy to the chest. The 504 patientsrepresent >90% of patients eligible for this study who underwentcoronary angiography during this period.

[0303] Angiographic analysis. Coronary angiograms were analyzed withhand-held calipers or visual analysis and divided into those revealingnormal coronaries (smooth arteries with either no stenosis or withstenosis ≦10%), mild disease (coronary arteries with a reduction inluminal diameter between 10% and 50%), single vessel disease (≧50% in asingle coronary artery or its major branches), two vessel coronaryartery disease (≧50% lumenal diameter stenosis in two coronary arteries)and three vessel disease (≧50% lumenal diameter stenosis in threecoronary arteries). Angiograms were analyzed blinded to patients' riskfactors and genetic analyses.

[0304] Laboratory analyses. Apolipoprotein A₁, apolipoprotein B, Lp (a)and fibrinogen assays were performed on the COBAS MIRA system. Normalranges for these assays are apolipoprotein A1, 115-190 mg/dL;apolipoprotein B, 70-160 mg/dL; and Lp (a), 2.5-7.0 mg/dL; a normalrange of fibrinogen is not reported. Total plasma homocysteine wasmeasured.

[0305] Definitions. A family history of coronary disease was consideredto be present if a first degree relative of the patient that did notsmoke or have diabetes mellitus developed coronary disease when ≦55years of age. Hyperlipidemia was defined as a total cholesterol ≧250mg/dL or an LDL ≧150 mg/dL, or ongoing treatment with lipid-loweringagents in patients in whom pre-treatment lipid values were unknown.Angina and heart failure were classified according to the Canadian HeartAssociation and New York State classification schemes, respectively.

[0306] Statistical methods. Values are expressed as percentages and asmeans±one standard deviation. For odds ratios, 95% confidence intervalsare presented in parentheses.

[0307] In preliminary analyses to determine correlates of coronarydisease, chi-square tests and one-way ANOVAs were first performed totest the association of various traditional and emerging risk factors,as well as allelic variants among the IL-1 cluster genes, among patientswith no disease, mild disease, one-vessel disease, two-vessel disease,and three-vessel disease. Coronary artery disease was then reclassifiedto compare patients with no disease or mild disease to patients withone-, two-, or three-vessel stenosis. Patients with some blockage butwith coronary stenosis <50% were considered to have mild coronarydisease and were grouped with those patients with no blockage (nodisease), while patients with stenosis ≧50% in one, two, or threecoronary arteries were grouped together for further analysis since thesepatients were considered to have a significant degree of coronary arterystenosis. The exact test for trends was used to test for trends in theproportion of patients with the polymorphisms.

[0308] Logistic regression models were fitted for the various riskfactors according to quartiles and tertiles with the odds ratiosreported for increasing quartile and tertile levels given. To analyzefurther the association of allelic variants of the IL-1 cluster geneswith coronary artery disease, multiple logistic regression models werefitted with statistically significant confounders included in eachmodel. All traditional and emerging risk factors were considered forinclusion in the model, and the model was fitted in a stepwise fashionto obtain the best fitting model where all factors included in the modelwere statistically significant. In addition, potential effect modifierswere considered for inclusion in the model. The response for themultiple logistic regression models was the presence or absence ofsignificant coronary artery stenosis defined above.

[0309] In addition to analyzing all subjects included in the study,further statistical analyses were performed on subjects ≦£60 years ofage and subjects >60 years of age separately. The analysis by age wasconsidered to be appropriate since age has been shown to be a strongrisk factor for coronary artery disease, and because genetic influencesin multifactorial diseases are believed to be most evident in earlyonset cases. In addition, since epistasis may determine that geneticinfluences have different outcomes on males and females, subset analysesby gender was also considered to be important and males and femalestreated separately in some of the analyses.

Example 6

[0310] The Munich Study

[0311] Methods

[0312] Patients

[0313] The study included 1850 consecutive Caucasian patients withsymptomatic coronary artery disease who underwent coronary stentimplantation at Deutsches Herzzentrum München and 1. Medizinische Klinikrechts der Isar der Technischen Universität München. All patients werescheduled for angiographic follow-up at 6 months. All patientsparticipating in this study gave written informed consent for theintervention, follow-up angiography, and genotype determination. Thestudy protocol conformed to the Declaration of Helsinki and was approvedby the institutional ethics committee. TABLE 1 Baseline clinicalcharacteristics. IL-IRN 1/2 or 2/2 (n = 896) IL-IRN 1/1 (n = 954) PAge-yr 63.4 ± 10.0 62.6 ± 10.0 0.11 Women-% 22.4 19.9 0.19 Arterialhypertension-% 67.2 68.9 0.44 Diabetes-% 22.7 19.4 0.08 Current orformer smoker-% 38.7 41.2 0.28 Elevated total cholesterol-% 42.5 43.10.81 Acute myocardial infarction-% 20.3 20.2 0.97 Unstable angina-% 27.927.8 0.95 Prior bypass surgery-% 10.6 11.5 0.53 Reduced left ventricularfunction-% 31.3 27.7 0.09 Number of diseased coronary vessels 0.39 -1vessel-% 29.2 27.3 -2 vessels-% 32.9 31.9 -3 vessels-% 37.8 40.9Periprocedural abciximab therapy-% 19.8 19.6 0.93

[0314] The protocol of stent placement and poststenting therapy isfamiliar to practitioners in the arts. Most of the stents were implantedhand-mounted on conventional angioplasty balloons. Postproceduraltherapy consisted of aspirin (100 mg twice daily, indefinitely) andticlopidine (250 mg twice daily for 4 weeks). Patients with suboptimalresults due to residual thrombus or dissection with flow impairmentafter stent implantation received additional therapy with abciximabgiven as bolus injection during stent insertion procedure and as a12-hours continuous infusion thereafter. The decision to give abciximabwas taken at the operator's discretion.

[0315] Determination of the IL-1RN Genotype

[0316] Genomic DNA was extracted from 200 ml of peripheral bloodleukocytes with the QIAamp Blood Kit (Qiagen, Hilden, Germany) and theHigh Pure PCR Template Preparation Kit (Boehringer Mannheim, Mannheim,Germany).

[0317] IL-1RN genotyping was performed with the ABI Prism SequenceDetection System (PE Applied Biosystems, Weiterstadt, Germany). The useof allele-specific fluorogenic probes in the 5′ nuclease reactioncombines DNA amplification and genotype determination into a singleassay 33. IL-1RN (+2018), a single base pair polymorphism in exon 2, wasthe polymorphism typed for this study 26. The nucleotide sequences ofprimers and probes were as follows: forward primer 5′ GGG ATG TTA ACCAGA AGA CCT TCT ATC T 3′, reverse primer 5′ CAA CCA CTC ACC TTC TAA ATTGAC ATT 3′, allele 1 probe 5′ AAC AAC CAA CTA GTT GCT GGA TAC TTG CAA3′, allele 2 probe 5′ ACA ACC AAC TAG TTG CCG GAT ACT TGC 3′. The probesfor allele 1 were labeled with the fluorescent dye 6-carboxy-fluorescein(FAM) and for allele 2 with the fluorescent dyetetrachloro-6-carboxy-fluorescein (TET) at the 5′ end. Both probes werelabeled with the quencher 6-carboxy-tetramethyl-rhodamine (TAMRA) attheir 3′ ends. The thermocycling protocol consisted of 40 cycles ofdenaturation at 95 C for 15 seconds and annealing/extension at 64 C for1 minute. Genotype validation was performed by repeating thedetermination in 20% of the patients using a duplicate DNA sample with anovel subject code unrelated to the original subject code. There was a100% matching between the 2 results.

[0318] Angiographic Assessment

[0319] Coronary lesions were classified according to the modifiedAmerican College of Cardiology/American Heart Association gradingsystem. Left ventricular function was assessed qualitatively on thebasis of biplane angiograms using a 7 segment division; the diagnosis ofreduced left ventricular function was established in the presence of atleast two hypokinetic segments in the contrast angiogram. Quantitativecomputer-assisted angiographic analysis was performed off-line onangiograms obtained just before stenting, immediately after stenting,and at follow up using the automated edge-detection system CMS (MedisMedical Imaging Systems, Nuenen, The Netherlands). Operators wereunaware of the patient's IL-1RN genotype. Identical projections of thetarget lesion were used for all assessed angiograms. Minimal lumendiameter, interpolated reference diameter, diameter stenosis, lesionlength and diameter of the maximally inflated balloon were theangiographic parameters obtained with this analysis system. Acute lumengain was calculated as the difference between minimal lumen diameter atthe end of intervention and minimal lumen diameter before theintervention. Late lumen loss was calculated as the difference betweenminimal lumen diameter at the end of intervention and minimal lumendiameter at the time of follow-up angiography. Loss index was calculatedas the ratio between late lumen loss and acute lumen gain.

[0320] Definitions and Study Endpoints

[0321] Primary endpoint of the study was restenosis. Two measures ofrestenosis were assessed: the incidence of angiographic restenosisdefined as a diameter stenosis of 50% at 6-month follow-up angiography,and the need for target vessel revascularization (PTCA or aortocoronarybypass surgery [CABG]) due to symptoms or signs of ischemia in thepresence of angiographic restenosis at the stented site over 1 yearafter the intervention. Other major adverse events evaluated were: deathfrom any cause and myocardial infarction. All deaths were considered dueto cardiac causes unless an autopsy established a noncardiac cause. Thediagnosis of acute myocardial infarction was based on the criteriaapplied in the EPISTENT trial (new pathological Q waves or a value ofcreatine kinase [CK] or its MB isoenzyme at least 3 times the upperlimit) 35. CK was determined systematically over the 48 hours followingstenting procedure. Clinical events were monitored throughout the 1-yearfollow-up period. The assessment was made on the basis of theinformation provided by hospital readmission records, referringphysician or phone interview with the patient. For all those patientswho revealed cardiac symptoms during the interview, at least oneclinical and electrocardiographic check-up was performed at theoutpatient clinic or by the referring physician.

[0322] Statistical Analysis

[0323] Discrete variables are expressed as counts or percentages andcompared with Chi-square or Fisher's exact test, as appropriate.Continuous variables are expressed as mean SD and compared by means ofthe unpaired, two-sided t-test or analysis of variance for more than 2groups. Risk analysis was performed calculating the odds ratio and the95% confidence interval. The main analysis consisted in comparingcombined heterozygous and homozygous carriers of the IL-11RN*2 allelewith homozygous carriers of the IL-11RN*1 allele. Moreover, theassociation between IL-1RN genotype and restenosis was assessed in amultivariate logistic regression model including also those clinical andlesion-related characteristics for which the comparison between carriersand noncarriers of the IL-1RN*2 allele showed a P-value 0.30. In thismultivariate model, we tested for the possible interaction betweenIL-1RN genotype and age. Since the relative contribution of geneticfactors to multifactorial processes such as restenosis may decrease withthe age, we carried out an additional analysis for a prespecifiedsubgroup of patients <60 years. Successively, we used test for trend forassessing gene dose effect, i.e. a stepwise increasing phenotypicresponse with the presence of 0, 1 or 2 putative alleles. Statisticalsignificance was accepted for P-values 0.05.

[0324] Results

[0325] Patients Characteristics

[0326] The observed IL-1RN genotypes in the study population were 1/1 in954 (51.6%), 1/2 in 742 (40.1%) and 2/2 in 154 (8.3%). Thus, allele 2frequency was 0.28. The observed distribution complied withHardy-Weinberg equilibrium. Main baseline characteristics of thepatients are listed in Table 1 and compared between carriers andnoncarriers of the IL-1RN*2 allele. There was a trend to a higherfrequency of diabetes and reduced left ventricular function amongcarriers of the IL-1RN*2 allele. The other characteristics were evenlydistributed between the 2 groups. The angiographic and proceduralcharacteristics at the time of intervention are listed in Table 2 andshow no significant differences between carriers and noncarriers of theIL-1RN*2 allele. TABLE 2 Lesion and procedural characteristics at thetime of intervention. IL-1RN 1/2 or 2/2 (n = 896) IL-1RN 1/1 (n = 954)Target coronary vessels 0.89 Left main-%  1.3  1.6 LAD-% 40.1 39.3 LCx-%19.9 20.0 RCA-% 32.6 31.9 Venous bypass graft-%  6.1  7.2 Complexlesions-% 75.2 74.1 0.58 Restenotic lesions-% 25.3 23.3 0.30 Beforestenting Reference diameter, mm 3.02 ± 0.53 3.05 ± 0.54 0.29 Diameterstenosis-% 79.1 ± 14.9 78.7 ± 15.7 0.57 Lesion length-mm 12.1 ± 6.9 12.1 ± 6.6  0.98 Procedural data Measured balloon diameter-mm 3.2 ± 0.53.2 ± 5   0.45 Maximal balloon pressure-atm 13.9 ± 3.3  13.8 ± 3.2  0.20Stented segment length-mm 20.0 ± 14.3 20.3 ± 13.6 0.70 Immediately afterstenting Diameter stenosis- 5.2 ± 9.1 5.4 ± 7.6 0.47

[0327] LAD indicates left anterior descending coronary artery; LCx, leftcircumflex coronary artery; RCA, right coronary artery; complex lesionswere defined as ACC/AHA lesion types B2 and C, according to the AmericanCollege of Cardiology/American Heart Association grading system.

[0328] IL-1ra Polymorphism, Mortality and Myocardial Infarction AfterStenting

[0329] Table 3 shows the adverse clinical events observed within thefirst 30 days after coronary stenting in carriers and noncarriers of theIL-1RN*2 allele. There was no association between the presence of theIL-1RN*2 allele and death, myocardial infarction or target vesselrevascularization, showing no significant influence of the polymorphismin the IL-1ra gene in the risk for early thrombotic events aftercoronary stenting. TABLE 3 Incidence of adverse events recorded duringthe early 30 days IL-1RN 1/2 or 2/2 (n = 896)  IL-1RN 1/1 (n = 954)Death- % 0.9 0.9 0.91 Nonfatal myocardial infarction- % 3.3 2.6 0.52-Q-wave- % 1.1 0.7 0.39 -non-Q-wave- % 2.2 1.9 0.60 Target vesselrevascularization- % 3.0 2.3 0.34

[0330] One-year follow-up indicated also that there is no correlationbetween the presence of the IL-1RN*2 allele and mortality or incidenceof myocardial infarction after the intervention. During the 1-yearperiod, mortality rate was 2.8% in the combined group of IL-1RN 1/2 andIL-1RN 2/2 patients and 2.2% in IL-1 1/1 patients (P=0.42), yielding anodds ratio of 1.28 (95% confidence interval, 0.71-2.29). The incidenceof nonfatal myocardial infarction was 3.5% in IL-1RN*2 allele carriersand 3.9% in homozygous carriers of the IL-1RN*1 allele (P=0.54), and therespective odds ratio was 0.86 (0.53-1.4).

[0331] IL-1ra Polymorphism and Restenosis After Stenting

[0332] Control angiography was performed in 84% of the patients after amedian of 188 days (interquartile range, 171-205 days). The proportionof patients with control angiography was similar in the 2 groups definedby the presence or absence of the IL-1RN*2 allele. Table 4 lists theresults of the quantitative assessment of 6-month angiograms. TABLE 4Results at follow-up angiography. IL-1RN 1/2 or 2/2 (n = 758) IL-1RN 1/1(n = 798) Late lumen loss-mm  1.160.82  1.240.86 0.07 Loss index 0.530.38  0.590.45 0.009 Diameter stenosis-% 41.826.2 45.228.7 0.015Restenosis rate- 30.2 35.6 0.024

[0333] Of note, loss index which reflects the hyperplastic responseafter stenting was significantly lower in patients who carried theIL-1RN*2 allele. The incidence of angiographic restenosis was alsosignificantly lower in carriers of the IL-1RN*2 allele, with 30.2% vs.35.6% in patients of the IL-1RN 1/1 genotype. Thus, the presence of theIL-1RN*2 allele was associated with a 22% decrease in restenosis rate(odds ratio, 0.78 [0.63-0.97]). Clinical restenosis expressed as theneed for target vessel revascularization was also significantly lower,with 17.7% in IL-1RN*2 allele carriers vs. 22.7% in homozygous patientsfor the IL-1RN*1 allele (P=0.026), yielding an odds ratio of 0.73(0.58-0.92).

[0334] Age, gender, the presence or absence of diabetes, smoking habit,reduced left ventricular function and restenotic lesions, vessel size(all variables differing in univariate analysis by a P-value 0.30) wereentered into the multivariate model for angiographic restenosis alongwith the presence or absence of the IL-1RN*2 allele. Older age(P=0.005), the presence of diabetes (P<0.001), restenotic lesion(P<0.001) and small vessel size (P<0.001) were independently correlatedwith an increased risk of restenosis. On the opposite, the presence ofthe IL-1RN*2 allele was independently (P<0.001) correlated with adecreased risk for restenosis with an adjusted odds ratio of 0.81(0.71-0.92). In addition, there was a significant interaction betweenthe presence of the IL-1RN*2 allele and age (P=0.009) as reflected by aprogressively stronger protective effect of this allele in youngerpatients.

[0335] The results of the analysis in the prespecified subgroup ofpatients <60 years (n=696) are presented in Table 5. During the 1-yearfollow-up period, 17.1% of the IL-1RN*2 allele carriers and 24.9% of thehomozygous IL-1RN*1 allele carriers needed target vesselrevascularization (P=0.013). Thus, the presence of the IL-1RN*2 allelewas associated with a 37% reduction (odds ratio: 0.63 [0.43-0.91]) ofthe need of ischemia-driven reinterventions. Quantitative angiographicdata obtained for the control study at 6 months (performed in 590 or 85%of patients <60 years) are displayed in Table 5. TABLE 5 Results atfollow-up angiography in patients < 60 years. IL-1RN 1/2 or 2/2 (n =273) IL-1RN 1/1 (n = 317) P Late lumen loss-mm  1.080.77  1.270.93 0.008Loss index  0.490.35  0.590.48 0.003 Diameter stenosis-% 39.324.146.730.5 0.001 Restenosis rate-% 25.6 38.5 <0.001  

[0336] The incidence of angiographic restenosis was 25.6% in thecombined group of IL-1RN 1/2 and IL-1RN 2/2 patients and 38.5% amongIL-1RN 1/1 patients (P<0.001), which corresponds to a 45% reduction(odds ratio: 0.55 [0.39-0.78]). The incidence of restenosis decreasedprogressively with heterozygosity and homozygosity for the IL-1RN*2allele. The rate of angiographic restenosis was 38.5% in IL-1RN 1/1patients, 26.3% in IL-1RN 1/2 patients and 22.4% in IL-1RN 2/2 patients(P=0.001, test for trend). The target vessel revascularization rate was24.9% in IL-1RN 1/1 patients, 17.9% in IL-1RN 1/2 patients and 13.2% inIL-1RN 2/2 patients (P=0.01, test for trend).

1 18 1 17 DNA Artificial Sequence Description of Artificial Sequenceprimer 1 ctcagcaaca ctcctat 17 2 17 DNA Artificial Sequence Descriptionof Artificial Sequence primer 2 tcctggtctg caggtaa 17 3 27 DNAArtificial Sequence Description of Artificial Sequence primer 3ctatctgagg aacaaccaac tagtagc 27 4 24 DNA Artificial SequenceDescription of Artificial Sequence primer 4 taggacattg cacctagggt ttgt24 5 20 DNA Artificial Sequence Description of Artificial Sequenceprimer 5 tggcattgat ctggttcatc 20 6 20 DNA Artificial SequenceDescription of Artificial Sequence primer 6 gtttaggaat cttcccactt 20 725 DNA Artificial Sequence Description of Artificial Sequence primer 7ctcaggtgtc ctcgaagaaa tcaaa 25 8 21 DNA Artificial Sequence Descriptionof Artificial Sequence primer 8 gcttttttgc tgtgagtccc g 21 9 30 DNAArtificial Sequence Description of Artificial Sequence primer 9atggttttag aaatcatcaa gcctagggca 30 10 30 DNA Artificial SequenceDescription of Artificial Sequence primer 10 aatgaaagga ggggaggatgacagaaatgt 30 11 28 DNA Artificial Sequence Description of ArtificialSequence primer 11 gggatgttaa ccagaagacc ttctatct 28 12 27 DNAArtificial Sequence Description of Artificial Sequence primer 12caaccactca ccttctaaat tgacatt 27 13 30 DNA Artificial SequenceDescription of Artificial Sequence primer 13 aacaaccaac tagttgctggatacttgcaa 30 14 25 DNA Artificial Sequence Description of ArtificialSequence primer 14 tgtacctaag cccacccttt agagc 25 15 20 DNA ArtificialSequence Description of Artificial Sequence primer 15 tggcctccagaaacctccaa 20 16 20 DNA Artificial Sequence Description of ArtificialSequence primer 16 gctgatattc tggtgggaaa 20 17 20 DNA ArtificialSequence Description of Artificial Sequence primer 17 ggcaagagcaaaactctgtc 20 18 27 DNA Artificial Sequence Description of ArtificialSequence primer 18 acaaccaact agttgccgga tacttgc 27

What is claimed is:
 1. A method for determining whether a patient has acardiovascular disorder, comprising: detecting a first cardiovasculardisorder associated allele in a nucleic acid sample from the patient,wherein detection of the first cardiovascular disorder associated alleleindicates that the patient has the cardiovascular disorder.
 2. Themethod of claim 1, wherein the first cardiovascular disorder associatedallele is selected from the group consisting of allele 2 of IL-1A(+4845), allele 2 of IL-1B (+3954), allele 1 of IL-1B (−511), allele 1of IL-1RN (+2018), and an allele in linkage disequilibrium with anaforementioned allele.
 3. The method of claim 1, wherein the firstcardiovascular disorder associated allele is selected from the groupconsisting of allele 1 of IL-1A (+4845), allele 1 of IL-1B (+3954),allele 2 of IL-1B (−511), allele 2 of IL-1RN (+2018), and an allele inlinkage disequilibrium with an aforementioned allele.
 4. The method ofclaim 1, wherein the first cardiovascular disorder associated allele isselected from the group consisting of allele 1 of IL-1A (+4845), allele1 of IL-1B (+3954), allele 1 of IL-1B (−511), allele 1 of IL-1RN(+2018), and an allele in linkage disequilibrium with an aforementionedallele.
 5. The method of claim 1, further comprising detecting a secondcardiovascular disorder associated allele in the nucleic acid sample,wherein detection of the second cardiovascular disorder associatedallele indicates that the patient has the cardiovascular disorder. 6.The method of claim 5, wherein the first cardiovascular disorderassociated allele is selected from the group consisting of allele 2 ofIL-1A (+4845), allele 2 of IL-1B (+3954), an allele in linkagedisequilibrium with allele 2 of IL-1A (+4845), and an allele in linkagedisequilibrium with allele 2 of IL-1B (+3954), and wherein the secondcardiovascular disorder associated allele is selected from the groupconsisting of allele 1 of IL-1B (−511), allele 1 of IL-1RN (+2018), anallele in linkage disequilibrium with allele 1 of IL-1B (−511), and anallele in linkage disequilibrium with allele 1 of IL-1RN (+2018).
 7. Themethod of claim 5, wherein the first cardiovascular disorder associatedallele is selected from the group consisting of allele 1 of IL-1A(+4845), allele 1 of IL-1B (+3954), an allele in linkage disequilibriumwith allele 1 of IL-1A (+4845), and an allele in linkage disequilibriumwith allele 1 of IL-1B (+3954), and wherein the second cardiovasculardisorder associated allele is selected from the group consisting ofallele 2 of IL-1B (−511), allele 2 of IL-1RN (+2018), an allele inlinkage disequilibrium with allele 2 of IL-1B (−511), and an allele inlinkage disequilibrium with allele 2 of IL-1RN (+2018).
 8. The method ofclaim 5, wherein the first cardiovascular disorder associated allele isselected from the group consisting of allele 1 of IL-1A (+4845), allele1 of IL-1B (+3954), an allele in linkage disequilibrium with allele 1 ofIL-1A (+4845), and an allele in linkage disequilibrium with allele 1 ofIL-1B (+3954), and wherein the second cardiovascular disorder associatedallele is selected from the group consisting of allele 1 of IL-1B(−511), allele 1 of IL-1RN (+2018), an allele in linkage disequilibriumwith allele 1 of IL-1B (−511), and an allele in linkage disequilibriumwith allele 1 of IL-1RN (+2018).
 9. The method of claim 1, wherein saiddetecting step is selected from the group consisting of: a) allelespecific oligonucleotide hybridization; b) size analysis; c) sequencing;d) hybridization; e) 5′ nuclease digestion; f) single-strandedconformation polymorphism; g) allele specific hybridization; h) primerspecific extension; and j) oligonucleotide ligation assay.
 10. Themethod of claim 1, further comprising amplifying the nucleic acidsample.
 11. The method of claim 10, wherein amplifying the nucleic acidsample employs a primer pair selected from the group consisting of anyof SEQ ID Nos. 1 and 2; 3 and 4; 5 and 6; 7 and 8; and 9 and
 10. 12. Themethod of claim 9, wherein said size analysis is preceded by arestriction enzyme digestion.
 13. The method of claim 12, wherein saidrestriction enzyme digestion uses a restriction enzyme selected from thegroup consisting of Alu I, Msp I, Nco I, Fnu 4HI, Ava I, Bsu 36 I, andTaq I.
 14. A kit for determining a presence of a cardiovascular disorderin a patient, comprising: a means for detecting an allele ofIL-1A(+4845), an allele of IL-1B(+3954), an allele of IL-1B(−511), anallele of IL-1RN(+2018), and an allele in linkage disequilibrium withaforesaid alleles; and a first primer oligonucleotide that hybridizes 5′or 3′ to an allele selected from the group consisting of an allele ofIL-1A (+4845), an allele of IL-1B (+3954), an allele of IL-1B(−511), anallele of IL-1RN(+2018), and an allele in linkage disequilibrium withaforesaid alleles.
 15. The kit of claim 14, further comprising a secondprimer oligonucleotide that hybridizes 5′ or 3′ to an allele selectedfrom the group consisting of an allele of IL-1A (+4845), an allele ofIL-1B (+3954), an allele of IL-1B(−511), an allele of IL-1RN(+2018), andan allele in linkage disequilibrium with aforesaid alleles.
 16. The kitof claim 14, which additionally comprises an amplifying primeroligonucleotide that hybridizes either 3′ or 5′ respectively to theallele for amplifying said allele.
 17. The kit of claim 16, wherein saidfirst primer, said second primer and said amplifying primeroligonucleotides hybridize to a region in the range of between about 50and about 1000 base pairs.
 18. The kit of claim 16, wherein said firstprimer, said second primer and said amplifying primer nucleotides areselected from the group consisting of any of SEQ ID Nos. 1-10.
 19. Thekit of claim 14, wherein the detection means is selected from the groupconsisting of: a) allele specific oligonucleotide hybridization; b) sizeanalysis; c) sequencing; d) hybridization; e) 5′ nuclease digestion; f)single-stranded conformation polymorphism; g) allele specifichybridization; h) primer specific extension; and j) oligonucleotideligation assay.
 20. The kit of claim 14, further comprising anamplification means.
 21. The kit of claim 14, further comprising acontrol.
 22. The method for treating a patient, comprising: detectingwhether the patient has a cardiovascular disorder associated allele,diagnosing a cardiovascular disorder, selecting a cardiovasculardisorder therapeutic, and providing the cardiovascular disordertherapeutic to the patient.
 23. The method of claim 22, wherein thecardiovascular disorder comprises a fragile plaque disorder.
 24. Themethod of claim 22, wherein the cardiovascular disorder comprises anocclusive disorder.
 25. The method of claim 22, wherein thecardiovascular disorder comprises an in-stent restenosis.
 26. The methodof claim 22, wherein the cardiovascular disorder further comprises acardiovascular disorder causing mutation that is in linkagedisequilibrium with the cardiovascular disorder associated allele. 27.The method of claim 22, further comprising identifying a presence of arisk factor for the cardiovascular disorder, and formulating a treatmentplan that reduces an effect of the risk factor on the patient.
 28. Themethod of claim 27, wherein identifying the presence of a risk factorcomprises performing a diagnostic test.
 29. The method of claim 27,wherein the treatment plan comprises an administration of a therapeuticagent that modifies the risk factor.
 30. The method of claim 22, whereinsaid detecting is performed using a technique selected from the groupconsisting of: a) allele specific oligonucleotide hybridization; b) sizeanalysis; c) sequencing; d) hybridization; e) 5′ nuclease digestion; f)single-stranded conformation polymorphism; g) allele specifichybridization; h) primer specific extension; and j) oligonucleotideligation assay.
 31. The method of claim 22, wherein the nucleic acidsample is subjected to an amplification step.
 32. The method of claim31, wherein said amplification step employs a primer selected from thegroup consisting of SEQ ID Nos. 1-10.
 33. The method of claim 30,wherein said size analysis is preceded by a restriction enzymedigestion.
 34. The method of claim 33, wherein said restriction enzymedigestion uses a restriction enzyme selected from the group consistingof Alu I, Msp I, Nco I, Fnu 4HI, Ava I, Bsu 36 I, and Taq I.
 35. Themethod of claim 22, wherein the cardiovascular disorder therapeuticcomprises a modulator of an IL-1 activity.
 36. The method of claim 35,wherein the IL-1 activity is IL-1α.
 37. A method of claim 35, whereinthe IL-1 activity is IL-1α.
 38. A method of claim 35, wherein the IL-1activity is IL-1RN.
 39. A method of claim 35, wherein the modulator isan IL-1 agonist.
 40. A method of claim 35, wherein the modulator is anIL-1 antagonist.